Difference: MarkGirvin (1 vs. 16)

• Name: Mark Girvin

• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: AECOMOffice
• Country: USA
• Phone: 718-430-2724

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E.

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E.

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin

• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: AECOMOffice
• Country: USA
• Phone: 718-430-2724
• Comment: Albert Einstein College of Medicine 303 Forchheimer, 1300 Morris Park Ave., Bronx, NY 10461 Biochemistry Department girvin@aecom.yu.edu FAX:(718) 430-8565

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of the ATP Synthase Membrane Domain

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@icar.bioc.aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: AECOMOffice
• Country: USA

• Comment: Albert Einstein College of Medicine 303 Forchheimer, 1300 Morris Park Ave., Bronx, NY 10461 Biochemistry Department girvin@aecom.yu.edu FAX:(718) 430-8565

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of the ATP Synthase Membrane Domain

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@icar.bioc.aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: AECOMOffice
• Country: USA
• Phone: 718-430-2025

• Horizontal size of text edit box:
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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of the ATP Synthase Membrane Domain

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@icar.bioc.aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: AECOMOffice
• Country: USA
• Phone: 718-430-2025

• Horizontal size of text edit box:
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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of the ATP Synthase Membrane Domain

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@icar.bioc.aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: AECOMOffice
• Country: USA

• Comment: 303 Forchheimer, 1200 Morris Park Ave., Bronx, NY 10461

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of the ATP Synthase Membrane Domain

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@icar.bioc.aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: AECOMOffice
• Country: USA
• Comment: 303 Forchheimer, 1200 Morris Park Ave., Bronx, NY 10461

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of the ATP Synthase Membrane Domain

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@icar.bioc.aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/

• Country: USA

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of the ATP Synthase Membrane Domain

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@icar.bioc.aecom.yu.edu

• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: 303 Forchheimer, 1200 Morris Park Ave., Bronx, NY 10461
• Country: USA
• Comment:

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of the ATP Synthase Membrane Domain

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin

• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: 303 Forchheimer, 1200 Morris Park Ave., Bronx, NY 10461
• Country: USA
• Comment:

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

Grant Number: 5R01GM055371-08 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of the ATP Synthase Membrane Domain

Abstract: DESCRIPTION (provided by applicant): The F1F0 ATP synthase is responsible for synthesizing the vast majority of cellular AlP^?. Not surprisingly, deleterious mutations in genes of the ATP synthase lead to inherited disorders, especially of nerves and muscles. The enzyme consists of two subcomplexes. The water-soluble F1 contains the catalytic sites for AlP^? synthesis and hydrolysis. The transmembrane F0 is responsible for proton transport. Remarkable progress has been made in understanding the structural and mechanistic aspects of catalysis by F1. As is always the case with membrane proteins, progress with the Fo has been much slower. Fo comprises three types of subunits in an a1b2c10 stoichiometry. Proton translocation through Fo is hypothesized to occur at the interface between the a-and c-subunits, beginning with a half-channel in subunit-a, moving through the essential Asp6l of subunit-c, and concluding with a second half-channel in subunit-a. Recent structures of subunit-c monomers in both protonation state suggest that during proton translocation the C-terminal helix of subunit-c rotates against subunit-a as a small "gear," driving rotation of a ring of c-subunits relative to the static subunits, and ultimately leading to the catalytic conformational changes in F1. This hypothesis will be tested by: 1) solving the structure of subunit-c in its oligomeric form, 2) determining the active site configurations of the c-subunits during the steps of proton translocation, 3) identifying the proton translocation pathways in subunit-a, and 4) determining how access of the active site Asp61 residue of subunit-c is limited to one side of the membrane at a time. The sample conditions and NMR methodologies developed to accomplish these aims will have general application to studying membrane protein structure by NMR. Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, few such structures are available.

Thesaurus Terms: conformation, hydrogen transporting ATP synthase, model design /development, protein folding, protein structure function, structural biology, structural model acidity /alkalinity, active site, chemical bond resonance, dimer, molecular dynamics, monomer, protonation, thermodynamics Escherichia coli, affinity chromatography, artificial membrane, chimeric protein, computer simulation, crystallization, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, protein purification, stable isotope double label

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: 303 Forchheimer, 1200 Morris Park Ave., Bronx, NY 10461
• Country: USA
• Comment:

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: 303 Forchheimer, 1200 Morris Park Ave., Bronx, NY 10461
• Country: USA
• Comment:

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Grant Number: 1R01GM072085-01 PI Name: GIRVIN, MARK E. PI Email: girvin@aecom.yu.edu PI Title: ASSOCIATE PROFESSOR Project Title: Structural Analysis of Multidrug Transport

Abstract: DESCRIPTION (provided by applicant): Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100- 110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR.

Thesaurus Terms: Staphylococcus aureus, bacterial protein, conformation, multidrug resistance, protein structure function glutamate, inhibitor /antagonist, micelle, pharmacokinetics, protein kinase A, protonation nuclear magnetic resonance spectroscopy

Institution: YESHIVA UNIVERSITY

1. W 185TH ST NEW YORK, NY 10033

• Name: Mark Girvin
• Email: girvin@aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.bioc.aecom.yu.edu/labs/girvlab/
• Location: 303 Forchheimer, 1200 Morris Park Ave., Bronx, NY 10461
• Country: USA
• Comment:

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• Name: Mark Girvin
• Email: girvin@aecom.yu.edu
• Company Name: Albert Einstein College of Medicine

• Country: USA
• Comment:

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• Horizontal size of text edit box:
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• Name: Mark Girvin
• Email: girvin@aecom.yu.edu
• Company Name: Albert Einstein College of Medicine
• Company URL: http://www.aecom.yu.edu
• Location: AECOMOffice
• Country: USA
• Comment:

Personal Preferences (details in TWikiVariables)

• Horizontal size of text edit box:
  • Set EDITBOXWIDTH = 70
• Vertical size of text edit box:
  • Set EDITBOXHEIGHT = 17
• Style of text edit box. width: 99% for full window width (default), width: auto to disable.
  • Set EDITBOXSTYLE = width: 99%
• Optionally write protect your home page: (set it to your WikiName)
  • Set ALLOWTOPICCHANGE =

Related topics

• TWikiPreferences has site-level preferences of TWiki.
• WebPreferences has preferences of the TWiki.Main web.
• TWikiUsers has a list of other TWiki users.