This is a proposal to investigate the structure and characteristics of the transition state in the dimerization/folding reaction of the GCN4-p1 peptide. Most models describing the folding pathways of proteins involve formation of secondary structure as a key component. The observation of kinetic intermediates that have secondary structure has been taken as strong support for these proposals. However, in preliminary experiments presented by Dr. Sosnick, it would appear that the transition state in the GCN4-p1 folding reaction, which is an encounter complex between two peptides leading to the homodimer, is lacking in helical character and has conformational flexibility similar to that of the unfolded peptide. These surprising results need to be clarified and enlarged. It is crucial to understand what, if not secondary structure, does guide the initial folding steps, and what features of the transition state allow it to proceed in an energetically downhill manner to the folded structure. The importance of specific structural/energetic features in early pathway steps will be studied with the simple coiled coil zipper, which provides a simple platform on which substitutions can be made that focus on specific structural features. Residues will be substituted that alter stability of these features, and folding rates will be measured using stopped flow spectroscopy. If a structural feature is fully formed at a substituted site in the transition state, then the free energy of the transition state and the equilibrium native stability will be altered equally. The unfolding rate will not change, and only the folding will be affected. For example, to test for the presence of helix, exterior residues having altered helix propensity will be substituted. If helix is present at the rate limiting step, only folding rates will be affected. The capability of focusing on particular features is very important and potentially represents a major advance in our ability to dissect the role of particular structural features in kinetic folding. This strategy will be applied to the folding of both the dimeric and cross-linked versions of the zipper, to test the effects of proximity, collision frequency and chain topology on folding.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM055694-05
Application #
6181132
Study Section
Special Emphasis Panel (ZRG3-BBCA (01))
Program Officer
Wehrle, Janna P
Project Start
1996-08-01
Project End
2001-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
5
Fiscal Year
2000
Total Cost
$107,100
Indirect Cost
Name
University of Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Lian, Huada; Qin, Jian; Freed, Karl F (2018) Dielectric virial expansion of polarizable dipolar spheres. J Chem Phys 149:163332
Wang, Zongan; Jumper, John M; Wang, Sheng et al. (2018) A Membrane Burial Potential with H-Bonds and Applications to Curved Membranes and Fast Simulations. Biophys J 115:1872-1884
Skinner, John J; Wang, Sheng; Lee, Jiyoung et al. (2017) Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome. Proc Natl Acad Sci U S A 114:13453-13458
Riback, Joshua A; Katanski, Christopher D; Kear-Scott, Jamie L et al. (2017) Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response. Cell 168:1028-1040.e19
Haddadian, Esmael J; Zhang, Hao; Freed, Karl F et al. (2017) Comparative Study of the Collective Dynamics of Proteins and Inorganic Nanoparticles. Sci Rep 7:41671
Baxa, Michael C; Yu, Wookyung; Adhikari, Aashish N et al. (2015) Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar. Proc Natl Acad Sci U S A 112:8302-7
Watkins, Herschel M; Simon, Anna J; Sosnick, Tobin R et al. (2015) Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions. Proc Natl Acad Sci U S A 112:6631-6
Baxa, Michael C; Haddadian, Esmael J; Jumper, John M et al. (2014) Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations. Proc Natl Acad Sci U S A 111:15396-401
Adhikari, Aashish N; Freed, Karl F; Sosnick, Tobin R (2013) Simplified protein models: predicting folding pathways and structure using amino acid sequences. Phys Rev Lett 111:028103
Liu, Yufeng; Haddadian, Esmael; Sosnick, Tobin R et al. (2013) A novel implicit solvent model for simulating the molecular dynamics of RNA. Biophys J 105:1248-57

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