Proteins acquire their unique functions through specific folding of their linear polypeptide chains. Misfolding results in numerous diseases, such as cystic fibrosis and various neurodegenerative disorders. Although a great deal of work has been done on the investigation of how the secondary and tertiary structures of proteins are formed, and both experimental and theoretical techniques for studying protein folding are continually becoming more refined, a quantitative and predictive understanding of protein folding is still not attainable. There are still many fundamental questions as to how specific and nonspecific interactions determine the protein folding pathways, the native and nonnative structures, and thermally and kinetically accessible conformation substates, and on what range of timescales do particular folding events occur. Addressing these questions presents the need for further studies with time-resolved spectroscopic techniques that can provide the necessary time resolution and structure sensitivities. The principal objective of the proposed research is therefore to develop new spectroscopic methods and new conformation probes that can be used to generate detailed structure interpretations of the transient folding species and their dynamics over the time range of interest. A detailed set of experiments are planned to gain detailed insight into the understanding of various aspects of the folding problem, including the helix-coil transition, early folding events and intermediates, and the mechanism of beta-sheet formation. The technical goals are: (a) to develop a nanosecond temperature jump infrared spectrometer that can measure both transient kinetics at discrete frequencies and time-resolved spectra at discrete reaction times; (b) to develop a microsecond FTIR coupled continuous-flow mixing apparatus; (c) to explore novel isotope editing techniques to permit site specific conformation studies; (d) to introduce nitriles into various amino acids as infrared probes of protein folding, dynamics and interactions; (e) to study the helix-coil transition; (f) to study the beta-sheet formation in the WW domain and its interaction with peptides; (g) to study the folding and spontaneous fluctuation of single GFP molecules.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065978-04
Application #
6917116
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
2002-07-01
Project End
2007-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
4
Fiscal Year
2005
Total Cost
$251,868
Indirect Cost
Name
University of Pennsylvania
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Lin, Chun-Wei; Gai, Feng (2017) Microscopic nucleation and propagation rates of an alanine-based ?-helix. Phys Chem Chem Phys 19:5028-5036
Ding, Bei; Hilaire, Mary Rose; Gai, Feng (2016) Infrared and Fluorescence Assessment of Protein Dynamics: From Folding to Function. J Phys Chem B 120:5103-13
Abaskharon, Rachel M; Gai, Feng (2016) Meandering Down the Energy Landscape of Protein Folding: Are We There Yet? Biophys J 110:1924-32
Mukherjee, Debopreeti; Gai, Feng (2016) Exciton circular dichroism couplet arising from nitrile-derivatized aromatic residues as a structural probe of proteins. Anal Biochem 507:74-8
Abaskharon, Rachel M; Gai, Feng (2016) Direct measurement of the tryptophan-mediated photocleavage kinetics of a protein disulfide bond. Phys Chem Chem Phys 18:9602-7
Mintzer, Mary Rose; Troxler, Thomas; Gai, Feng (2015) p-Cyanophenylalanine and selenomethionine constitute a useful fluorophore-quencher pair for short distance measurements: application to polyproline peptides. Phys Chem Chem Phys 17:7881-7
Ma, Jianqiang; Pazos, Ileana M; Zhang, Wenkai et al. (2015) Site-specific infrared probes of proteins. Annu Rev Phys Chem 66:357-77
Hilaire, Mary Rose; Abaskharon, Rachel M; Gai, Feng (2015) Biomolecular Crowding Arising from Small Molecules, Molecular Constraints, Surface Packing, and Nano-Confinement. J Phys Chem Lett 6:2546-53
Pazos, Ileana M; Ahmed, Ismail A; Berríos, Mariana I León et al. (2015) Sensing pH via p-cyanophenylalanine fluorescence: Application to determine peptide pKa and membrane penetration kinetics. Anal Biochem 483:21-6
Oh, Kwang-Im; Smith-Dupont, Kathryn B; Markiewicz, Beatrice N et al. (2015) Kinetics of peptide folding in lipid membranes. Biopolymers 104:281-90

Showing the most recent 10 out of 72 publications