Large-amplitude conformational changes in proteins, including loop motions, relative motions between domains, collective "breathing" of protein cores, ligand-binding or oligomerization reactions, and overall folding-unfolding events, may be closely coupled, and in some instances rate-limiting, to biological functions such as molecular recognition, transitions along the catalytic cycle of enzymes, and inhibition or activation of proteins through intra-or inter-molecular protein-protein interactions. Mutations that perturb dynamical processes and conformational equilibria are associated with significant pathology, including loss or gain of function and misfolding. Recent developments, including those from the applicant laboratory, have opened new opportunities for investigation of large amplitude conformational dynamic processes on microsecond-millisecond time scales using NMR spin relaxation measurements at equilibrium in solution and with atomic site resolution, without potential complications introduced by non-native modifications necessary for other solution-state spectroscopic techniques. The proposed research has four primary objectives: (1) identification of the mechanistic basis for cell adhesion mediated by domain (strand) swapping in the cadherin superfamily, (2) assessment of the role of conformational mobility in catalysis by the essential enzyme ornithine 5'-monophosphate decarboxylase, (3) elucidation of the folding mechanism and description of the unfolded-state ensemble for the villin headpiece domain HP67, and (4) development of novel experimental and theoretical methods for characterizing protein dynamics on ?s-ms time scales. Time-dependent structural changes underlie the normal function of proteins, and misfunction in genetic diseases, cancer, and other pathologies;the proposed research will quantify this linkage for three protein systems involved in cellular structure and basic metabolism. Completion of these goals will enable additional future applications to a wide range of protein systems of biological importance.

Public Health Relevance

The present proposal addresses the coupling between structure, dynamics, and function in cell adhesion mediated by cadherins, catalysis by the essential enzyme orotidine 5'-monophosphate decarboxylase, and folding of the villin headpiece domain. Elaboration of the roles of conformational dynamics in actuating and regulating these processes is essential for understanding the fundamental functions of proteins in human health and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059273-15
Application #
8490392
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
1999-05-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
15
Fiscal Year
2013
Total Cost
$341,330
Indirect Cost
$128,432
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Sher, Inbal; Chang, Shih Chieh; Li, Ying et al. (2014) Conformational flexibility in the binding surface of the potassium channel blocker ShK. Chembiochem 15:2402-10
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Li, Ying; Rance, Mark; Palmer 3rd, Arthur G (2014) Rotation operator propagators for time-varying radiofrequency pulses in NMR spectroscopy: applications to shaped pulses and pulse trains. J Magn Reson 248:105-14
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Cho, Jae-Hyun; Muralidharan, Vasant; Vila-Perello, Miquel et al. (2011) Tuning protein autoinhibition by domain destabilization. Nat Struct Mol Biol 18:550-5
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Cho, Jae-Hyun; O'Connell, Nichole; Raleigh, Daniel P et al. (2010) Phi-value analysis for ultrafast folding proteins by NMR relaxation dispersion. J Am Chem Soc 132:450-1
Li, Ying; Palmer 3rd, Arthur G (2009) TROSY-selected ZZ-exchange experiment for characterizing slow chemical exchange in large proteins. J Biomol NMR 45:357-60
O'Connell, Nichole E; Grey, Michael J; Tang, Yuefeng et al. (2009) Partially folded equilibrium intermediate of the villin headpiece HP67 defined by 13C relaxation dispersion. J Biomol NMR 45:85-98

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