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Transition state heterogeneity in GCN4 coiled coil folding studied by using multisite mutations and crosslinking
L B Moran1, J P Schneider, A Kentsis
1Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
Summary
Protein folding pathways differ significantly based on chain topology. Dimeric proteins fold via multiple routes, while crosslinked versions follow a single pathway, highlighting the critical role of chain structure in protein folding dynamics.
Area of Science:
- Protein folding and biophysics
- Molecular biology and biochemistry
Background:
- The GCN4-p1 coiled coil is a 33-residue alpha-helical protein fragment derived from a transcriptional activator.
- Understanding protein folding pathways is crucial for deciphering protein function and dysfunction.
Purpose of the Study:
- To investigate the folding behavior of dimeric and covalently crosslinked GCN4-p1 coiled coils.
- To determine how chain topology influences protein folding routes and transition state ensembles.
Main Methods:
- Utilized multisite substitutions to probe folding mechanisms.
- Analyzed activation energies and intrinsic helical propensities.
- Compared folding pathways of dimeric versus crosslinked monomeric GCN4-p1.
Main Results:
- Dimeric GCN4-p1 folds via multiple routes with nucleation sites distributed throughout the protein.
- Minimal helical structure exists before chain collision; significant helical formation occurs in the postcollision transition state.
- Crosslinked GCN4-p1 folds along a single pathway with distinct transition state structuring based on proximity to the crosslink.
Conclusions:
- Protein folding routes and transition state heterogeneity are critically dependent on chain topology.
- The structural organization in the transition state ensemble varies significantly between dimeric and crosslinked GCN4-p1.
- Chain topology dictates the selection and characteristics of protein folding pathways.