Proteins must fold into specific three-dimensional structures to be functional, in a process dictated by their primary sequence. Current understanding of the mechanisms by which proteins fold is limited by the deceivingly simple picture arising from standard kinetic experiments. In these experiments, folding appears as a two or three-state process because the inter-conversions between the myriad of intermediate structures that determine the mechanism are too transient to be directly detected. In this proposal, a group of new experimental approaches to circumvent these limitation is presented. To facilitate extracting mechanistic information from kinetics observations, a catalog of small proteins with simple structural patterns; i.e., structural archetypes, will be produced. Their folding properties will be investigated by fast-kinetic methods such as the laser-induced temperature-jump technique. In an alternative approach, the existence of the theoretically predicted downhill scenario for folding will be explored experimentally. Identification of downhill folders is important because during downhill folding all intermediate structures are potentially detectable. Additionally, kinetic methods with improved structural and/or time resolution will be developed. A two-dimensional version of the phi-analysis will be implemented to investigate the population dynamics of transition-state ensembles for folding. Time-dependent information on transient intermediates will be obtained for the first time from equilibrium nuclear magnetic resonance hydrogen-exchange experiments by performing them in kinetic coupling mode. The application of these kinetic techniques to study the folding of structural archetypes and downhill folders will provide direct information about the structural rules governing protein folding.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066800-03
Application #
6891950
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
2003-05-02
Project End
2008-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
3
Fiscal Year
2005
Total Cost
$254,352
Indirect Cost
Name
University of Maryland College Park
Department
Chemistry
Type
Schools of Earth Sciences/Natur
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
Li, Peng; Oliva, Fabiana Y; Naganathan, Athi N et al. (2009) Dynamics of one-state downhill protein folding. Proc Natl Acad Sci U S A 106:103-8
Naganathan, Athi N; Munoz, Victor (2008) Determining denaturation midpoints in multiprobe equilibrium protein folding experiments. Biochemistry 47:6752-61
Fung, Adam; Li, Peng; Godoy-Ruiz, Raquel et al. (2008) Expanding the realm of ultrafast protein folding: gpW, a midsize natural single-domain with alpha+beta topology that folds downhill. J Am Chem Soc 130:7489-95
Halskau Jr, Oyvind; Perez-Jimenez, Raul; Ibarra-Molero, Beatriz et al. (2008) Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics. Proc Natl Acad Sci U S A 105:8625-30
Naganathan, Athi N; Doshi, Urmi; Munoz, Victor (2007) Protein folding kinetics: barrier effects in chemical and thermal denaturation experiments. J Am Chem Soc 129:5673-82
Munoz, Victor (2007) Conformational dynamics and ensembles in protein folding. Annu Rev Biophys Biomol Struct 36:395-412
Naganathan, Athi N; Doshi, Urmi; Fung, Adam et al. (2006) Dynamics, energetics, and structure in protein folding. Biochemistry 45:8466-75
Naganathan, Athi N; Sanchez-Ruiz, Jose M; Munoz, Victor (2005) Direct measurement of barrier heights in protein folding. J Am Chem Soc 127:17970-1
Naganathan, Athi N; Munoz, Victor (2005) Scaling of folding times with protein size. J Am Chem Soc 127:480-1
Akmal, Arya; Munoz, Victor (2004) The nature of the free energy barriers to two-state folding. Proteins 57:142-52

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