Difference: TomMuir (1 vs. 23)

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name:

• Country: USA

The New York Academy of Sciences has honored Rockefeller University professor Tom W. Muir with a Blavatnik Award for Young Scientists, and also presented Rockefeller president Paul Nurse with a Science and the City Award for Outstanding Accomplishments in New York City. The awards were given at the fifth annual Science and City Gala held last night at a Manhattan restaurant.

Muir, who is Richard E. Salomon Family Professor and head of Rockefeller’s Selma and Lawrence Ruben Laboratory of Synthetic Protein Chemistry, is one of three faculty winners of this year’s Blavatnik awards, chosen from among nine faculty finalists. He studies the physicochemical basis of protein function in complex systems of biomedical interest. His lab has combined the tools of organic chemistry, biochemistry and cell biology to develop new technologies that provide insight into how proteins work, research with wide potential for elucidating protein function in the postgenomic era.

Established in 2007 with funds from the Blavatnik Charitable Foundation, The Blavatnik Awards recognize the achievements of young scientists and engineers from New York, New Jersey and Connecticut who have contributed significantly to interdisciplinary research in the life sciences, physical sciences and engineering. The award carries an unrestricted cash prize of $25,000 for faculty winners. Three other Rockefeller University researchers were finalists in this year’s postdoctoral competition and Rockefeller’s Leslie B. Vosshall, the Chemers Family Associate Professor, was a winner in the 2007 competition.

Nurse is head of the Laboratory of Yeast Genetics and Cell Biology and has been president of The Rockefeller University since 2003. The Science and the City Award honors scientists for achievements that contribute to the continuation of New York City as a center of scientific excellence. Nurse shares the award with Herbert Pardes, president and CEO of NewYork^?-Presbyterian Hospital.

The New York Academy of Sciences (NYAS), founded in 1817, is one of the country’s oldest scientific institutions. With about 25,000 members in 140 countries, NYAS is an independent, nonprofit organization committed to advancing science, technology and society worldwide in an effort to positively impact major global challenges with science-based solutions and increase the number of scientifically informed individuals in society at large. This year’s gala raised more than $1 million for the organization.

Public Information on Grants associated with NYSBC Grant Number: 9R01GM086868-10 Project Title: Structure, Function and Applications of Inteins PI Information: Name Email Title MUIR, TOM W. muirt@rockefeller.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA^? (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFB

Abstract: A research program will be undertaken in the area of TGF-p signaling, a fundamental cellular process that is implicated in multiple human diseases, including cancer. A key feature of the signaling pathway is phosphorylation of a family of latent transcription factors termed R-Smads, a biochemical event that leads to nuclear accumulation. The central hypothesis of this research is that continuous nucleocytoplasmic shuttling of R-Smads with repeated cycles of receptor-mediated phosphorylation and nuclear dephosphorylation, permits constant sensing of the activation status of the receptor and hence efficient termination of signaling upon receptor inactivation. To explore this, we will study the three critical phases in the biochemical lifetime of the R-Smad, Smad2, namely, bis-phosphorylation of the protein by the activated TpR^?-l receptor, nuclear import of the phosphorylated Smad2/Smad4 complex, and nuclear dephosphorylation of Smad2 followed by its nuclear export. Key to this research program is our ability to introduce biochemical and biophysical probes site-specifically into phosphorylated forms of T[3R-I and Smad2, thereby allowing us to control and monitor their activities. Chemistry-driven protein engineering approaches will be used in conjunction with established biophysical and cell biological approaches to study the detailed mechanisms by which Smad2 interacts with the activated receptor complex, and thereafter shuttles to and from the nucleus. The specific aims are: 1. To Study the Mechanisms Underlying R-Smad Activation: We will use biochemical and structural techniques to investigate how activated TpR^?-l receptor recognizes and then double phosphorylates Smad2. 2. To Study the Mechanisms Underlying R-Smad Nuclear Import: We will use biochemical and cell biological techniques to study the molecular mechanisms by which phosphorylation of Smad2 leads to its nuclear accumulation. 3. To Study the Mechanisms Underlying R-Smad Nuclear Export. We will identify and characterize the putative nuclear phosphatase responsible for removing the activation phosphates from Smad2. By providing a more complete quantitative and structural understanding of the basic signaling pathway, this research program will lay a firmer foundation for the rational design of small molecular therapies based on manipulation of TGF-psignaling.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms: phosphorylation, receptor base, cell, chemistry, family, human, identity, lead, motivation, neoplasm /cancer, nuclear receptor, phosphate, protein, protein engineering, therapy, transcription factor

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2009 Department: SYNTHETIC PROTEIN CHEMISTRY LAB Project Start: 01-APR-1997 Project End: 31-MAR-2010 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: SBCA

• Name: Tom Muir
• Email: muirt@rockefeller.edu

• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121

The New York Academy of Sciences has honored Rockefeller University professor Tom W. Muir with a Blavatnik Award for Young Scientists, and also presented Rockefeller president Paul Nurse with a Science and the City Award for Outstanding Accomplishments in New York City. The awards were given at the fifth annual Science and City Gala held last night at a Manhattan restaurant.

Muir, who is Richard E. Salomon Family Professor and head of Rockefeller’s Selma and Lawrence Ruben Laboratory of Synthetic Protein Chemistry, is one of three faculty winners of this year’s Blavatnik awards, chosen from among nine faculty finalists. He studies the physicochemical basis of protein function in complex systems of biomedical interest. His lab has combined the tools of organic chemistry, biochemistry and cell biology to develop new technologies that provide insight into how proteins work, research with wide potential for elucidating protein function in the postgenomic era.

Established in 2007 with funds from the Blavatnik Charitable Foundation, The Blavatnik Awards recognize the achievements of young scientists and engineers from New York, New Jersey and Connecticut who have contributed significantly to interdisciplinary research in the life sciences, physical sciences and engineering. The award carries an unrestricted cash prize of $25,000 for faculty winners. Three other Rockefeller University researchers were finalists in this year’s postdoctoral competition and Rockefeller’s Leslie B. Vosshall, the Chemers Family Associate Professor, was a winner in the 2007 competition.

Nurse is head of the Laboratory of Yeast Genetics and Cell Biology and has been president of The Rockefeller University since 2003. The Science and the City Award honors scientists for achievements that contribute to the continuation of New York City as a center of scientific excellence. Nurse shares the award with Herbert Pardes, president and CEO of NewYork^?-Presbyterian Hospital.

The New York Academy of Sciences (NYAS), founded in 1817, is one of the country’s oldest scientific institutions. With about 25,000 members in 140 countries, NYAS is an independent, nonprofit organization committed to advancing science, technology and society worldwide in an effort to positively impact major global challenges with science-based solutions and increase the number of scientifically informed individuals in society at large. This year’s gala raised more than $1 million for the organization.

Public Information on Grants associated with NYSBC Grant Number: 9R01GM086868-10 Project Title: Structure, Function and Applications of Inteins PI Information: Name Email Title MUIR, TOM W. muirt@rockefeller.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA^? (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFB

Abstract: A research program will be undertaken in the area of TGF-p signaling, a fundamental cellular process that is implicated in multiple human diseases, including cancer. A key feature of the signaling pathway is phosphorylation of a family of latent transcription factors termed R-Smads, a biochemical event that leads to nuclear accumulation. The central hypothesis of this research is that continuous nucleocytoplasmic shuttling of R-Smads with repeated cycles of receptor-mediated phosphorylation and nuclear dephosphorylation, permits constant sensing of the activation status of the receptor and hence efficient termination of signaling upon receptor inactivation. To explore this, we will study the three critical phases in the biochemical lifetime of the R-Smad, Smad2, namely, bis-phosphorylation of the protein by the activated TpR^?-l receptor, nuclear import of the phosphorylated Smad2/Smad4 complex, and nuclear dephosphorylation of Smad2 followed by its nuclear export. Key to this research program is our ability to introduce biochemical and biophysical probes site-specifically into phosphorylated forms of T[3R-I and Smad2, thereby allowing us to control and monitor their activities. Chemistry-driven protein engineering approaches will be used in conjunction with established biophysical and cell biological approaches to study the detailed mechanisms by which Smad2 interacts with the activated receptor complex, and thereafter shuttles to and from the nucleus. The specific aims are: 1. To Study the Mechanisms Underlying R-Smad Activation: We will use biochemical and structural techniques to investigate how activated TpR^?-l receptor recognizes and then double phosphorylates Smad2. 2. To Study the Mechanisms Underlying R-Smad Nuclear Import: We will use biochemical and cell biological techniques to study the molecular mechanisms by which phosphorylation of Smad2 leads to its nuclear accumulation. 3. To Study the Mechanisms Underlying R-Smad Nuclear Export. We will identify and characterize the putative nuclear phosphatase responsible for removing the activation phosphates from Smad2. By providing a more complete quantitative and structural understanding of the basic signaling pathway, this research program will lay a firmer foundation for the rational design of small molecular therapies based on manipulation of TGF-psignaling.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms: phosphorylation, receptor base, cell, chemistry, family, human, identity, lead, motivation, neoplasm /cancer, nuclear receptor, phosphate, protein, protein engineering, therapy, transcription factor

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2009 Department: SYNTHETIC PROTEIN CHEMISTRY LAB Project Start: 01-APR-1997 Project End: 31-MAR-2010 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: SBCA

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA +12123277368
• Comment: Board Member

The New York Academy of Sciences has honored Rockefeller University professor Tom W. Muir with a Blavatnik Award for Young Scientists, and also presented Rockefeller president Paul Nurse with a Science and the City Award for Outstanding Accomplishments in New York City. The awards were given at the fifth annual Science and City Gala held last night at a Manhattan restaurant.

Muir, who is Richard E. Salomon Family Professor and head of Rockefeller’s Selma and Lawrence Ruben Laboratory of Synthetic Protein Chemistry, is one of three faculty winners of this year’s Blavatnik awards, chosen from among nine faculty finalists. He studies the physicochemical basis of protein function in complex systems of biomedical interest. His lab has combined the tools of organic chemistry, biochemistry and cell biology to develop new technologies that provide insight into how proteins work, research with wide potential for elucidating protein function in the postgenomic era.

Established in 2007 with funds from the Blavatnik Charitable Foundation, The Blavatnik Awards recognize the achievements of young scientists and engineers from New York, New Jersey and Connecticut who have contributed significantly to interdisciplinary research in the life sciences, physical sciences and engineering. The award carries an unrestricted cash prize of $25,000 for faculty winners. Three other Rockefeller University researchers were finalists in this year’s postdoctoral competition and Rockefeller’s Leslie B. Vosshall, the Chemers Family Associate Professor, was a winner in the 2007 competition.

Nurse is head of the Laboratory of Yeast Genetics and Cell Biology and has been president of The Rockefeller University since 2003. The Science and the City Award honors scientists for achievements that contribute to the continuation of New York City as a center of scientific excellence. Nurse shares the award with Herbert Pardes, president and CEO of NewYork^?-Presbyterian Hospital.

The New York Academy of Sciences (NYAS), founded in 1817, is one of the country’s oldest scientific institutions. With about 25,000 members in 140 countries, NYAS is an independent, nonprofit organization committed to advancing science, technology and society worldwide in an effort to positively impact major global challenges with science-based solutions and increase the number of scientifically informed individuals in society at large. This year’s gala raised more than $1 million for the organization.

Public Information on Grants associated with NYSBC Grant Number: 9R01GM086868-10 Project Title: Structure, Function and Applications of Inteins PI Information: Name Email Title MUIR, TOM W. muirt@rockefeller.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA^? (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFB

Abstract: A research program will be undertaken in the area of TGF-p signaling, a fundamental cellular process that is implicated in multiple human diseases, including cancer. A key feature of the signaling pathway is phosphorylation of a family of latent transcription factors termed R-Smads, a biochemical event that leads to nuclear accumulation. The central hypothesis of this research is that continuous nucleocytoplasmic shuttling of R-Smads with repeated cycles of receptor-mediated phosphorylation and nuclear dephosphorylation, permits constant sensing of the activation status of the receptor and hence efficient termination of signaling upon receptor inactivation. To explore this, we will study the three critical phases in the biochemical lifetime of the R-Smad, Smad2, namely, bis-phosphorylation of the protein by the activated TpR^?-l receptor, nuclear import of the phosphorylated Smad2/Smad4 complex, and nuclear dephosphorylation of Smad2 followed by its nuclear export. Key to this research program is our ability to introduce biochemical and biophysical probes site-specifically into phosphorylated forms of T[3R-I and Smad2, thereby allowing us to control and monitor their activities. Chemistry-driven protein engineering approaches will be used in conjunction with established biophysical and cell biological approaches to study the detailed mechanisms by which Smad2 interacts with the activated receptor complex, and thereafter shuttles to and from the nucleus. The specific aims are: 1. To Study the Mechanisms Underlying R-Smad Activation: We will use biochemical and structural techniques to investigate how activated TpR^?-l receptor recognizes and then double phosphorylates Smad2. 2. To Study the Mechanisms Underlying R-Smad Nuclear Import: We will use biochemical and cell biological techniques to study the molecular mechanisms by which phosphorylation of Smad2 leads to its nuclear accumulation. 3. To Study the Mechanisms Underlying R-Smad Nuclear Export. We will identify and characterize the putative nuclear phosphatase responsible for removing the activation phosphates from Smad2. By providing a more complete quantitative and structural understanding of the basic signaling pathway, this research program will lay a firmer foundation for the rational design of small molecular therapies based on manipulation of TGF-psignaling.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms: phosphorylation, receptor base, cell, chemistry, family, human, identity, lead, motivation, neoplasm /cancer, nuclear receptor, phosphate, protein, protein engineering, therapy, transcription factor

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2009 Department: SYNTHETIC PROTEIN CHEMISTRY LAB Project Start: 01-APR-1997 Project End: 31-MAR-2010 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: SBCA

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA +12123277368
• Comment: Board Member

The New York Academy of Sciences has honored Rockefeller University professor Tom W. Muir with a Blavatnik Award for Young Scientists, and also presented Rockefeller president Paul Nurse with a Science and the City Award for Outstanding Accomplishments in New York City. The awards were given at the fifth annual Science and City Gala held last night at a Manhattan restaurant.

Muir, who is Richard E. Salomon Family Professor and head of Rockefeller’s Selma and Lawrence Ruben Laboratory of Synthetic Protein Chemistry, is one of three faculty winners of this year’s Blavatnik awards, chosen from among nine faculty finalists. He studies the physicochemical basis of protein function in complex systems of biomedical interest. His lab has combined the tools of organic chemistry, biochemistry and cell biology to develop new technologies that provide insight into how proteins work, research with wide potential for elucidating protein function in the postgenomic era.

Established in 2007 with funds from the Blavatnik Charitable Foundation, The Blavatnik Awards recognize the achievements of young scientists and engineers from New York, New Jersey and Connecticut who have contributed significantly to interdisciplinary research in the life sciences, physical sciences and engineering. The award carries an unrestricted cash prize of $25,000 for faculty winners. Three other Rockefeller University researchers were finalists in this year’s postdoctoral competition and Rockefeller’s Leslie B. Vosshall, the Chemers Family Associate Professor, was a winner in the 2007 competition.

Nurse is head of the Laboratory of Yeast Genetics and Cell Biology and has been president of The Rockefeller University since 2003. The Science and the City Award honors scientists for achievements that contribute to the continuation of New York City as a center of scientific excellence. Nurse shares the award with Herbert Pardes, president and CEO of NewYork^?-Presbyterian Hospital.

The New York Academy of Sciences (NYAS), founded in 1817, is one of the country’s oldest scientific institutions. With about 25,000 members in 140 countries, NYAS is an independent, nonprofit organization committed to advancing science, technology and society worldwide in an effort to positively impact major global challenges with science-based solutions and increase the number of scientifically informed individuals in society at large. This year’s gala raised more than $1 million for the organization.

Public Information on Grants associated with NYSBC Grant Number: 9R01GM086868-10 Project Title: Structure, Function and Applications of Inteins PI Information: Name Email Title MUIR, TOM W. muirt@rockefeller.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA^? (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFB

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA +12123277368
• Comment: Board Member

The New York Academy of Sciences has honored Rockefeller University professor Tom W. Muir with a Blavatnik Award for Young Scientists, and also presented Rockefeller president Paul Nurse with a Science and the City Award for Outstanding Accomplishments in New York City. The awards were given at the fifth annual Science and City Gala held last night at a Manhattan restaurant.

Muir, who is Richard E. Salomon Family Professor and head of Rockefeller’s Selma and Lawrence Ruben Laboratory of Synthetic Protein Chemistry, is one of three faculty winners of this year’s Blavatnik awards, chosen from among nine faculty finalists. He studies the physicochemical basis of protein function in complex systems of biomedical interest. His lab has combined the tools of organic chemistry, biochemistry and cell biology to develop new technologies that provide insight into how proteins work, research with wide potential for elucidating protein function in the postgenomic era.

Established in 2007 with funds from the Blavatnik Charitable Foundation, The Blavatnik Awards recognize the achievements of young scientists and engineers from New York, New Jersey and Connecticut who have contributed significantly to interdisciplinary research in the life sciences, physical sciences and engineering. The award carries an unrestricted cash prize of $25,000 for faculty winners. Three other Rockefeller University researchers were finalists in this year’s postdoctoral competition and Rockefeller’s Leslie B. Vosshall, the Chemers Family Associate Professor, was a winner in the 2007 competition.

Nurse is head of the Laboratory of Yeast Genetics and Cell Biology and has been president of The Rockefeller University since 2003. The Science and the City Award honors scientists for achievements that contribute to the continuation of New York City as a center of scientific excellence. Nurse shares the award with Herbert Pardes, president and CEO of NewYork^?-Presbyterian Hospital.

The New York Academy of Sciences (NYAS), founded in 1817, is one of the country’s oldest scientific institutions. With about 25,000 members in 140 countries, NYAS is an independent, nonprofit organization committed to advancing science, technology and society worldwide in an effort to positively impact major global challenges with science-based solutions and increase the number of scientifically informed individuals in society at large. This year’s gala raised more than $1 million for the organization.

Public Information on Grants associated with NYSBC Grant Number: 9R01GM086868-10 Project Title: Structure, Function and Applications of Inteins PI Information: Name Email Title MUIR, TOM W. muirt@rockefeller.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA^? (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFB

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA +12123277368
• Comment: Board Member

The New York Academy of Sciences has honored Rockefeller University professor Tom W. Muir with a Blavatnik Award for Young Scientists, and also presented Rockefeller president Paul Nurse with a Science and the City Award for Outstanding Accomplishments in New York City. The awards were given at the fifth annual Science and City Gala held last night at a Manhattan restaurant.

Muir, who is Richard E. Salomon Family Professor and head of Rockefeller’s Selma and Lawrence Ruben Laboratory of Synthetic Protein Chemistry, is one of three faculty winners of this year’s Blavatnik awards, chosen from among nine faculty finalists. He studies the physicochemical basis of protein function in complex systems of biomedical interest. His lab has combined the tools of organic chemistry, biochemistry and cell biology to develop new technologies that provide insight into how proteins work, research with wide potential for elucidating protein function in the postgenomic era.

Established in 2007 with funds from the Blavatnik Charitable Foundation, The Blavatnik Awards recognize the achievements of young scientists and engineers from New York, New Jersey and Connecticut who have contributed significantly to interdisciplinary research in the life sciences, physical sciences and engineering. The award carries an unrestricted cash prize of $25,000 for faculty winners. Three other Rockefeller University researchers were finalists in this year’s postdoctoral competition and Rockefeller’s Leslie B. Vosshall, the Chemers Family Associate Professor, was a winner in the 2007 competition.

Nurse is head of the Laboratory of Yeast Genetics and Cell Biology and has been president of The Rockefeller University since 2003. The Science and the City Award honors scientists for achievements that contribute to the continuation of New York City as a center of scientific excellence. Nurse shares the award with Herbert Pardes, president and CEO of NewYork^?-Presbyterian Hospital.

The New York Academy of Sciences (NYAS), founded in 1817, is one of the country’s oldest scientific institutions. With about 25,000 members in 140 countries, NYAS is an independent, nonprofit organization committed to advancing science, technology and society worldwide in an effort to positively impact major global challenges with science-based solutions and increase the number of scientifically informed individuals in society at large. This year’s gala raised more than $1 million for the organization.

Public Information on Grants associated with NYSBC Grant Number: 9R01GM086868-10 Project Title: Structure, Function and Applications of Inteins PI Information: Name Email Title MUIR, TOM W. muirt@rockefeller.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA^? (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFB

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA +12123277368
• Comment: Board Member

The New York Academy of Sciences has honored Rockefeller University professor Tom W. Muir with a Blavatnik Award for Young Scientists, and also presented Rockefeller president Paul Nurse with a Science and the City Award for Outstanding Accomplishments in New York City. The awards were given at the fifth annual Science and City Gala held last night at a Manhattan restaurant.

Muir, who is Richard E. Salomon Family Professor and head of Rockefeller’s Selma and Lawrence Ruben Laboratory of Synthetic Protein Chemistry, is one of three faculty winners of this year’s Blavatnik awards, chosen from among nine faculty finalists. He studies the physicochemical basis of protein function in complex systems of biomedical interest. His lab has combined the tools of organic chemistry, biochemistry and cell biology to develop new technologies that provide insight into how proteins work, research with wide potential for elucidating protein function in the postgenomic era.

Established in 2007 with funds from the Blavatnik Charitable Foundation, The Blavatnik Awards recognize the achievements of young scientists and engineers from New York, New Jersey and Connecticut who have contributed significantly to interdisciplinary research in the life sciences, physical sciences and engineering. The award carries an unrestricted cash prize of $25,000 for faculty winners. Three other Rockefeller University researchers were finalists in this year’s postdoctoral competition and Rockefeller’s Leslie B. Vosshall, the Chemers Family Associate Professor, was a winner in the 2007 competition.

Nurse is head of the Laboratory of Yeast Genetics and Cell Biology and has been president of The Rockefeller University since 2003. The Science and the City Award honors scientists for achievements that contribute to the continuation of New York City as a center of scientific excellence. Nurse shares the award with Herbert Pardes, president and CEO of NewYork^?-Presbyterian Hospital.

The New York Academy of Sciences (NYAS), founded in 1817, is one of the country’s oldest scientific institutions. With about 25,000 members in 140 countries, NYAS is an independent, nonprofit organization committed to advancing science, technology and society worldwide in an effort to positively impact major global challenges with science-based solutions and increase the number of scientifically informed individuals in society at large. This year’s gala raised more than $1 million for the organization.

Public Information on Grants associated with NYSBC Grant Number: 9R01GM086868-10 Project Title: Structure, Function and Applications of Inteins PI Information: Name Email Title MUIR, TOM W. muirt@rockefeller.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA^? (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFB

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA +12123277368
• Comment: Board Member

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA^? (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFB

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice

• Comment: Board Member

Public Information on Grants associated with NYSBC Grant Number: 9R01GM086868-10 Project Title: Structure, Function and Applications of Inteins PI Information: Name Email Title MUIR, TOM W. muirt@rockefeller.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA^? (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFB

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA
• Comment: Board Member

Public Information on Grants associated with NYSBC

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA
• Comment: Board Member

Public Information on Grants associated with NYSBC

Grant Number: 5R01EB001991-06 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Segmental Labeling for Protein Structure by NMR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications.

Thesaurus Terms: nuclear magnetic resonance spectroscopy, protein structure, stable isotope double label, structural biology, technology /technique development esterification, recombinant protein, thioester bioimaging /biomedical imaging

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2004 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 30-JUN-2008 ICD: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING IRG: ZRG1

Grant Number: 5R01GM055843-09 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Regulation and Folding of a Multidomain Adaptor Protein

Abstract: DESCRIPTION: (provided by applicant) A research program will be undertaken to study the structure, function and regulation of the adaptor protein, cCrk-ll, which plays an important role in cellular signaling events such as proliferation, differentiation, cell adhesion and cytoskeletal reorganization. There is mounting evidence, including our own preliminary data, for significant structural and functional interplay between the individual modular domains in this proto-oncogene. These intramolecular interactions appear to be important for the normal regulation of this molecule. Indeed, mutant forms of the Crk protein (as well as the viral oncogene, v-Crk) lacking various autoregulatory domains are known to transform cells. To better understand the molecular mechanisms of autoregulation, we will use protein engineering approaches, newly developed in our laboratory, to study the effect of native context on the stability, folding kinetics, backbone dynamics and function of the three component Src Homology Domains in the context of full-length c-Crk-fl. Key to this research program is the protein semi-synthesis technology, expressed protein ligation, which allows biochemical and biophysical probes to be site-specifically introduced into large proteins. Our studies will also provide much needed structural and functional information on the poorly understood C-terminal third of c-Crk-ll molecule, a region that we hypothesize regulates the function of the protein through intramolecular interactions. Lastly, we propose to develop photochemical strategies to control the regulatory state of c-Crk-ll with light. Specifically, we will prepare semi-synthetic c-Crk-H analogs containing photocaged phosphorylated amino acid derivatives at position Tyr22 1. Proof ofprinciple studies will then be performed to explore whether we can photochemically trigger phosphorylation of Y 21 in c-Crk-II, thereby controlling the regulatory state of the protein. Ultimately, this methodology will allow the controlled and synchronized release of active phospho-protein into cells with second-range kinetic resolution. The precise temporal and spatial control of delivery will provide information on the localization, trafficking, and metabolism of the target proteins. In summary, many biomedically important proteins, including a large number of oncogenes, contain multiple protein modules. There is pressing need to develop approaches that provide a way of extracting detailed structural and functional information on a specific domain within the context of a large multi-domain protein. The approaches developed in this proposal will be generally applicable to these types of structural and biochemical problems.

Thesaurus Terms: binding protein, protein binding, protein folding, protein kinase, protein structure function, protooncogene combinatorial chemistry, phosphorylation, photochemistry, protein localization, protein transport, synthetic enzyme

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-APR-1997 Project End: 31-MAR-2006 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BNP

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA
• Comment: Board Member

Grant Number: 5R01EB001991-06 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Segmental Labeling for Protein Structure by NMR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications.

Thesaurus Terms: nuclear magnetic resonance spectroscopy, protein structure, stable isotope double label, structural biology, technology /technique development esterification, recombinant protein, thioester bioimaging /biomedical imaging

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2004 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 30-JUN-2008 ICD: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING IRG: ZRG1

Grant Number: 5R01GM055843-09 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Regulation and Folding of a Multidomain Adaptor Protein

Abstract: DESCRIPTION: (provided by applicant) A research program will be undertaken to study the structure, function and regulation of the adaptor protein, cCrk-ll, which plays an important role in cellular signaling events such as proliferation, differentiation, cell adhesion and cytoskeletal reorganization. There is mounting evidence, including our own preliminary data, for significant structural and functional interplay between the individual modular domains in this proto-oncogene. These intramolecular interactions appear to be important for the normal regulation of this molecule. Indeed, mutant forms of the Crk protein (as well as the viral oncogene, v-Crk) lacking various autoregulatory domains are known to transform cells. To better understand the molecular mechanisms of autoregulation, we will use protein engineering approaches, newly developed in our laboratory, to study the effect of native context on the stability, folding kinetics, backbone dynamics and function of the three component Src Homology Domains in the context of full-length c-Crk-fl. Key to this research program is the protein semi-synthesis technology, expressed protein ligation, which allows biochemical and biophysical probes to be site-specifically introduced into large proteins. Our studies will also provide much needed structural and functional information on the poorly understood C-terminal third of c-Crk-ll molecule, a region that we hypothesize regulates the function of the protein through intramolecular interactions. Lastly, we propose to develop photochemical strategies to control the regulatory state of c-Crk-ll with light. Specifically, we will prepare semi-synthetic c-Crk-H analogs containing photocaged phosphorylated amino acid derivatives at position Tyr22 1. Proof ofprinciple studies will then be performed to explore whether we can photochemically trigger phosphorylation of Y 21 in c-Crk-II, thereby controlling the regulatory state of the protein. Ultimately, this methodology will allow the controlled and synchronized release of active phospho-protein into cells with second-range kinetic resolution. The precise temporal and spatial control of delivery will provide information on the localization, trafficking, and metabolism of the target proteins. In summary, many biomedically important proteins, including a large number of oncogenes, contain multiple protein modules. There is pressing need to develop approaches that provide a way of extracting detailed structural and functional information on a specific domain within the context of a large multi-domain protein. The approaches developed in this proposal will be generally applicable to these types of structural and biochemical problems.

Thesaurus Terms: binding protein, protein binding, protein folding, protein kinase, protein structure function, protooncogene combinatorial chemistry, phosphorylation, photochemistry, protein localization, protein transport, synthetic enzyme

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-APR-1997 Project End: 31-MAR-2006 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BNP

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA
• Comment: Board Member

Public Information on Grants associated with NYSBC

Grant Number: 5R01EB001991-06 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Segmental Labeling for Protein Structure by NMR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications.

Thesaurus Terms: nuclear magnetic resonance spectroscopy, protein structure, stable isotope double label, structural biology, technology /technique development esterification, recombinant protein, thioester bioimaging /biomedical imaging

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2004 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 30-JUN-2008 ICD: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING IRG: ZRG1

Grant Number: 5R01GM055843-09 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Regulation and Folding of a Multidomain Adaptor Protein

Abstract: DESCRIPTION: (provided by applicant) A research program will be undertaken to study the structure, function and regulation of the adaptor protein, cCrk-ll, which plays an important role in cellular signaling events such as proliferation, differentiation, cell adhesion and cytoskeletal reorganization. There is mounting evidence, including our own preliminary data, for significant structural and functional interplay between the individual modular domains in this proto-oncogene. These intramolecular interactions appear to be important for the normal regulation of this molecule. Indeed, mutant forms of the Crk protein (as well as the viral oncogene, v-Crk) lacking various autoregulatory domains are known to transform cells. To better understand the molecular mechanisms of autoregulation, we will use protein engineering approaches, newly developed in our laboratory, to study the effect of native context on the stability, folding kinetics, backbone dynamics and function of the three component Src Homology Domains in the context of full-length c-Crk-fl. Key to this research program is the protein semi-synthesis technology, expressed protein ligation, which allows biochemical and biophysical probes to be site-specifically introduced into large proteins. Our studies will also provide much needed structural and functional information on the poorly understood C-terminal third of c-Crk-ll molecule, a region that we hypothesize regulates the function of the protein through intramolecular interactions. Lastly, we propose to develop photochemical strategies to control the regulatory state of c-Crk-ll with light. Specifically, we will prepare semi-synthetic c-Crk-H analogs containing photocaged phosphorylated amino acid derivatives at position Tyr22 1. Proof ofprinciple studies will then be performed to explore whether we can photochemically trigger phosphorylation of Y 21 in c-Crk-II, thereby controlling the regulatory state of the protein. Ultimately, this methodology will allow the controlled and synchronized release of active phospho-protein into cells with second-range kinetic resolution. The precise temporal and spatial control of delivery will provide information on the localization, trafficking, and metabolism of the target proteins. In summary, many biomedically important proteins, including a large number of oncogenes, contain multiple protein modules. There is pressing need to develop approaches that provide a way of extracting detailed structural and functional information on a specific domain within the context of a large multi-domain protein. The approaches developed in this proposal will be generally applicable to these types of structural and biochemical problems.

Thesaurus Terms: binding protein, protein binding, protein folding, protein kinase, protein structure function, protooncogene combinatorial chemistry, phosphorylation, photochemistry, protein localization, protein transport, synthetic enzyme

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-APR-1997 Project End: 31-MAR-2006 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BNP

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA
• Comment: Board Member

Public Information on Grants associated with NYSBC

Grant Number: 5R01EB001991-06 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Segmental Labeling for Protein Structure by NMR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications.

Thesaurus Terms: nuclear magnetic resonance spectroscopy, protein structure, stable isotope double label, structural biology, technology /technique development esterification, recombinant protein, thioester bioimaging /biomedical imaging

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2004 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 30-JUN-2008 ICD: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING IRG: ZRG1

Grant Number: 5R01GM055843-09 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Regulation and Folding of a Multidomain Adaptor Protein

Abstract: DESCRIPTION: (provided by applicant) A research program will be undertaken to study the structure, function and regulation of the adaptor protein, cCrk-ll, which plays an important role in cellular signaling events such as proliferation, differentiation, cell adhesion and cytoskeletal reorganization. There is mounting evidence, including our own preliminary data, for significant structural and functional interplay between the individual modular domains in this proto-oncogene. These intramolecular interactions appear to be important for the normal regulation of this molecule. Indeed, mutant forms of the Crk protein (as well as the viral oncogene, v-Crk) lacking various autoregulatory domains are known to transform cells. To better understand the molecular mechanisms of autoregulation, we will use protein engineering approaches, newly developed in our laboratory, to study the effect of native context on the stability, folding kinetics, backbone dynamics and function of the three component Src Homology Domains in the context of full-length c-Crk-fl. Key to this research program is the protein semi-synthesis technology, expressed protein ligation, which allows biochemical and biophysical probes to be site-specifically introduced into large proteins. Our studies will also provide much needed structural and functional information on the poorly understood C-terminal third of c-Crk-ll molecule, a region that we hypothesize regulates the function of the protein through intramolecular interactions. Lastly, we propose to develop photochemical strategies to control the regulatory state of c-Crk-ll with light. Specifically, we will prepare semi-synthetic c-Crk-H analogs containing photocaged phosphorylated amino acid derivatives at position Tyr22 1. Proof ofprinciple studies will then be performed to explore whether we can photochemically trigger phosphorylation of Y 21 in c-Crk-II, thereby controlling the regulatory state of the protein. Ultimately, this methodology will allow the controlled and synchronized release of active phospho-protein into cells with second-range kinetic resolution. The precise temporal and spatial control of delivery will provide information on the localization, trafficking, and metabolism of the target proteins. In summary, many biomedically important proteins, including a large number of oncogenes, contain multiple protein modules. There is pressing need to develop approaches that provide a way of extracting detailed structural and functional information on a specific domain within the context of a large multi-domain protein. The approaches developed in this proposal will be generally applicable to these types of structural and biochemical problems.

Thesaurus Terms: binding protein, protein binding, protein folding, protein kinase, protein structure function, protooncogene combinatorial chemistry, phosphorylation, photochemistry, protein localization, protein transport, synthetic enzyme

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-APR-1997 Project End: 31-MAR-2006 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BNP

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA

Public Information on Grants associated with NYSBC

Grant Number: 5R01EB001991-06 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Segmental Labeling for Protein Structure by NMR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications.

Thesaurus Terms: nuclear magnetic resonance spectroscopy, protein structure, stable isotope double label, structural biology, technology /technique development esterification, recombinant protein, thioester bioimaging /biomedical imaging

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2004 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 30-JUN-2008 ICD: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING IRG: ZRG1

Grant Number: 5R01GM055843-09 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Regulation and Folding of a Multidomain Adaptor Protein

Abstract: DESCRIPTION: (provided by applicant) A research program will be undertaken to study the structure, function and regulation of the adaptor protein, cCrk-ll, which plays an important role in cellular signaling events such as proliferation, differentiation, cell adhesion and cytoskeletal reorganization. There is mounting evidence, including our own preliminary data, for significant structural and functional interplay between the individual modular domains in this proto-oncogene. These intramolecular interactions appear to be important for the normal regulation of this molecule. Indeed, mutant forms of the Crk protein (as well as the viral oncogene, v-Crk) lacking various autoregulatory domains are known to transform cells. To better understand the molecular mechanisms of autoregulation, we will use protein engineering approaches, newly developed in our laboratory, to study the effect of native context on the stability, folding kinetics, backbone dynamics and function of the three component Src Homology Domains in the context of full-length c-Crk-fl. Key to this research program is the protein semi-synthesis technology, expressed protein ligation, which allows biochemical and biophysical probes to be site-specifically introduced into large proteins. Our studies will also provide much needed structural and functional information on the poorly understood C-terminal third of c-Crk-ll molecule, a region that we hypothesize regulates the function of the protein through intramolecular interactions. Lastly, we propose to develop photochemical strategies to control the regulatory state of c-Crk-ll with light. Specifically, we will prepare semi-synthetic c-Crk-H analogs containing photocaged phosphorylated amino acid derivatives at position Tyr22 1. Proof ofprinciple studies will then be performed to explore whether we can photochemically trigger phosphorylation of Y 21 in c-Crk-II, thereby controlling the regulatory state of the protein. Ultimately, this methodology will allow the controlled and synchronized release of active phospho-protein into cells with second-range kinetic resolution. The precise temporal and spatial control of delivery will provide information on the localization, trafficking, and metabolism of the target proteins. In summary, many biomedically important proteins, including a large number of oncogenes, contain multiple protein modules. There is pressing need to develop approaches that provide a way of extracting detailed structural and functional information on a specific domain within the context of a large multi-domain protein. The approaches developed in this proposal will be generally applicable to these types of structural and biochemical problems.

Thesaurus Terms: binding protein, protein binding, protein folding, protein kinase, protein structure function, protooncogene combinatorial chemistry, phosphorylation, photochemistry, protein localization, protein transport, synthetic enzyme

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-APR-1997 Project End: 31-MAR-2006 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BNP

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA
• Comment: Board Member

Grant Number: 5R01EB001991-06 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Segmental Labeling for Protein Structure by NMR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications.

Thesaurus Terms: nuclear magnetic resonance spectroscopy, protein structure, stable isotope double label, structural biology, technology /technique development esterification, recombinant protein, thioester bioimaging /biomedical imaging

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2004 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 30-JUN-2008 ICD: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING IRG: ZRG1

Grant Number: 5R01GM055843-09 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Regulation and Folding of a Multidomain Adaptor Protein

Abstract: DESCRIPTION: (provided by applicant) A research program will be undertaken to study the structure, function and regulation of the adaptor protein, cCrk-ll, which plays an important role in cellular signaling events such as proliferation, differentiation, cell adhesion and cytoskeletal reorganization. There is mounting evidence, including our own preliminary data, for significant structural and functional interplay between the individual modular domains in this proto-oncogene. These intramolecular interactions appear to be important for the normal regulation of this molecule. Indeed, mutant forms of the Crk protein (as well as the viral oncogene, v-Crk) lacking various autoregulatory domains are known to transform cells. To better understand the molecular mechanisms of autoregulation, we will use protein engineering approaches, newly developed in our laboratory, to study the effect of native context on the stability, folding kinetics, backbone dynamics and function of the three component Src Homology Domains in the context of full-length c-Crk-fl. Key to this research program is the protein semi-synthesis technology, expressed protein ligation, which allows biochemical and biophysical probes to be site-specifically introduced into large proteins. Our studies will also provide much needed structural and functional information on the poorly understood C-terminal third of c-Crk-ll molecule, a region that we hypothesize regulates the function of the protein through intramolecular interactions. Lastly, we propose to develop photochemical strategies to control the regulatory state of c-Crk-ll with light. Specifically, we will prepare semi-synthetic c-Crk-H analogs containing photocaged phosphorylated amino acid derivatives at position Tyr22 1. Proof ofprinciple studies will then be performed to explore whether we can photochemically trigger phosphorylation of Y 21 in c-Crk-II, thereby controlling the regulatory state of the protein. Ultimately, this methodology will allow the controlled and synchronized release of active phospho-protein into cells with second-range kinetic resolution. The precise temporal and spatial control of delivery will provide information on the localization, trafficking, and metabolism of the target proteins. In summary, many biomedically important proteins, including a large number of oncogenes, contain multiple protein modules. There is pressing need to develop approaches that provide a way of extracting detailed structural and functional information on a specific domain within the context of a large multi-domain protein. The approaches developed in this proposal will be generally applicable to these types of structural and biochemical problems.

Thesaurus Terms: binding protein, protein binding, protein folding, protein kinase, protein structure function, protooncogene combinatorial chemistry, phosphorylation, photochemistry, protein localization, protein transport, synthetic enzyme

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-APR-1997 Project End: 31-MAR-2006 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BNP

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA

Grant Number: 5R01EB001991-06 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Segmental Labeling for Protein Structure by NMR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications.

Thesaurus Terms: nuclear magnetic resonance spectroscopy, protein structure, stable isotope double label, structural biology, technology /technique development esterification, recombinant protein, thioester bioimaging /biomedical imaging

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2004 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 30-JUN-2008 ICD: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING IRG: ZRG1

Abstract: DESCRIPTION: (provided by applicant) A research program will be undertaken to study the structure, function and regulation of the adaptor protein, cCrk-ll, which plays an important role in cellular signaling events such as proliferation, differentiation, cell adhesion and cytoskeletal reorganization. There is mounting evidence, including our own preliminary data, for significant structural and functional interplay between the individual modular domains in this proto-oncogene. These intramolecular interactions appear to be important for the normal regulation of this molecule. Indeed, mutant forms of the Crk protein (as well as the viral oncogene, v-Crk) lacking various autoregulatory domains are known to transform cells. To better understand the molecular mechanisms of autoregulation, we will use protein engineering approaches, newly developed in our laboratory, to study the effect of native context on the stability, folding kinetics, backbone dynamics and function of the three component Src Homology Domains in the context of full-length c-Crk-fl. Key to this research program is the protein semi-synthesis technology, expressed protein ligation, which allows biochemical and biophysical probes to be site-specifically introduced into large proteins. Our studies will also provide much needed structural and functional information on the poorly understood C-terminal third of c-Crk-ll molecule, a region that we hypothesize regulates the function of the protein through intramolecular interactions. Lastly, we propose to develop photochemical strategies to control the regulatory state of c-Crk-ll with light. Specifically, we will prepare semi-synthetic c-Crk-H analogs containing photocaged phosphorylated amino acid derivatives at position Tyr22 1. Proof ofprinciple studies will then be performed to explore whether we can photochemically trigger phosphorylation of Y 21 in c-Crk-II, thereby controlling the regulatory state of the protein. Ultimately, this methodology will allow the controlled and synchronized release of active phospho-protein into cells with second-range kinetic resolution. The precise temporal and spatial control of delivery will provide information on the localization, trafficking, and metabolism of the target proteins. In summary, many biomedically important proteins, including a large number of oncogenes, contain multiple protein modules. There is pressing need to develop approaches that provide a way of extracting detailed structural and functional information on a specific domain within the context of a large multi-domain protein. The approaches developed in this proposal will be generally applicable to these types of structural and biochemical problems.

Thesaurus Terms: binding protein, protein binding, protein folding, protein kinase, protein structure function, protooncogene combinatorial chemistry, phosphorylation, photochemistry, protein localization, protein transport, synthetic enzyme

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-APR-1997 Project End: 31-MAR-2006 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BNP

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA
• Comment:

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Public Information on Grants associated with NYSBC

Grant Number: 5R01EB001991-06 PI Name: MUIR, TOM W. PI Email: muirt@rockefeller.edu PI Title: PROFESSOR Project Title: Segmental Labeling for Protein Structure by NMR

Abstract: DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications.

Thesaurus Terms: nuclear magnetic resonance spectroscopy, protein structure, stable isotope double label, structural biology, technology /technique development esterification, recombinant protein, thioester bioimaging /biomedical imaging

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2004 Department: LAB/SYNTHETIC PROTEIN CHEM Project Start: 01-SEP-1999 Project End: 30-JUN-2008 ICD: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING IRG: ZRG1

• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA
• Comment:

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• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu/research/abstract.php?id=121
• Location: RockefellerUOffice
• Country: USA
• Comment:

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• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University

• Location: RockefellerUOffice
• Country: USA
• Comment:

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• Name: Tom Muir
• Email: muirt@rockefeller.edu
• Company Name: The Rockefeller University
• Company URL: http://www.rockefeller.edu
• Location: RockefellerUOffice
• Country: USA
• Comment:

Personal Preferences (details in TWikiVariables)

• Horizontal size of text edit box:
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  • Set EDITBOXHEIGHT = 17
• Style of text edit box. width: 99% for full window width (default), width: auto to disable.
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