Difference: AnnMcDermott (20 vs. 21)

• Name: Ann McDermott
• Email: aem5@columbia.edu
• Company Name: Columbia University
• Company URL: http://www.cise.columbia.edu/nsec/team/mcdermott.html
• Location: ColumbiaUOffice
• Country: USA
• Comment: (212)854-8393

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Abstract: DESCRIPTION (provided by applicant): Triosephosphate isomerase, an isomerase catalyzing an uphill reaction, offers a prime opportunity to stabilize and probe key kinetic and structural features of a Michaelis complex. This proposal capitalizes on our recent advances in stabilizing the Michaelis complex, and our optimization of a range of biophysical probes for the system, so that individual kinetic events and intermediate(s) can be studied. X-ray diffraction at 1.2Angstroms of the Michaelis complex shows a highly compressed active site with unusually short hydrogen bonds and an unexpected substrate torsional state. Recent data, utilizing three spectroscopic methods, indicate that loop opening and product release is rate determining for enzymatic throughput, and invite the hypothesis that loop opening may be triggered by the completion of the enzymatic reaction. The following issues are now ripe for pursuit. (1) Mutants in which the general base glutamic acid 165 and the general acid histidine 95 are replaced with alternative hydrogen bond partners are catalytically inactive. How does selection of the active site amino acids influence compression in the Michaelis complex and polarization of the substrate? (2) What are the chemical species in the Michaelis complex, and how do their concentrations depend upon temperature? Does the proposed enediolate chemical intermediate have a significant population, as might be expected based upon isotope washout? The substrate, while "pinned" through hydrogen bonds to the active site, appears to be mobile at the carbon centers, suggestive of an active rearrangement. Are the chemical reaction rates faster than the loop's motion, as suggested by the minimal primary isotope effects? (3) What are the conformational and ionization changes in the active site of the protein for the Michaelis complex, and what changes accompany the progress along the chemical reaction coordinate? How is the general base E165 positioned for the initial stage of the reaction? (4) What implications would changes in the ligand or mutations in the strongly conserved loop residues have for loop opening rates and for catalysis? These questions would be pursued through solid state NMR, vibrational spectroscopy and X-ray diffraction experiments, both static and dynamic .

Thesaurus Terms: active site, aminoacid, chemical kinetics, enzyme complex, triose phosphate isomerase catalyst, compression, conformation, fungal protein, gene mutation, solid state, temperature Raman spectrometry, X ray crystallography, infrared spectrometry, nuclear magnetic resonance spectroscopy, protein purification

Institution: COLUMBIA UNIV NEW YORK MORNINGSIDE 1210 AMSTERDAM AVE, MC 2205 NEW YORK, NY 10027 Fiscal Year: 2004 Department: CHEMISTRY Project Start: 01-AUG-2002 Project End: 31-JUL-2006 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BBCB

NSF Org MCB Intial Amendment Date July 28, 2003 Latest Amendment Date May 15, 2005 Award Number 0316248 Award Instrument Continuing grant Program Manager Kamal Shukla MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences Start Date July 15, 2003 Expires June 30, 2006 (Estimated) Awarded Amount to Date $477914 Investigator(s) AnnMcDermott aem5@columbia.edu(Principal Investigator) Sponsor Columbia University 1210 Amsterdam Avenue; MC 2205 New York, NY 10027 212/854-6851 NSF Program(s) BIOMOLECULAR SYSTEMS Field Application(s) Program Reference Code(s) BIOT,9183,1164 Program Element Code(s) 1144 Abstract

In this project the PI will develop new solid state NMR methods to study protein structure. In the previous grant period, the PI has largely completed the site-specific NMR resonance assignments for microcrystalline human Ubiquitin, providing proof of principle for the first stage in denovo structure determination by solid state NMR. In this project , the PI will confirm the existing assignments, and locate signals for the mobile portions. With the recent availability of high magnetic field strengths, her group intends to attempt more challenging and biologically interesting targets; two additional proteins would be assigned as solid phase precipitates: Calmodulin and Triosephosphate Isomerase. Finally, the PI will determine the 3D structure of Ubiquitin based on solid-state NMR data, utilizing several sources of experimental data. Torsional constraints are available from the chemical shifts themselves, which will be analyzed using statistical databases from solution NMR as well as from computed shifts. Hydrogen bond correlations will be measured via N-H...C correlations. Tertiary contacts will be measured using spin diffusion as well as selective recoupling experiments. Structures would be calculated using commercially available software. While structural genomics proceeds apace, many important non-soluble proteins are not readily characterized; the PI's laboratory will attempt characterization of some of the non-soluble targets when the methods are established through this work. The PI is involved with collaborations with several academic and industrial labs that will facilitate transfer of the information obtained through this research. The core effort, however, is the training of outstanding graduate students. High school and undergraduate students will be trained and exposed to structural biology and magnetic resonance in the PI's laboratory.