title: Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange.
abstract: The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λYA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 10(5) s(-1) and a stability of 7.4 kcal·mol(-1) at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational ϕ analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637;
authors.forename: Wookyung
authors.initials: W
authors.lastname: Yu
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637; Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637;
authors.forename: Michael C
authors.initials: MC
authors.lastname: Baxa
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637;
authors.forename: Isabelle
authors.initials: I
authors.lastname: Gagnon
authors.affiliation: Department of Chemistry, University of Chicago, Chicago, IL 60637; James Franck Institute, University of Chicago, Chicago, IL 60637; Computation Institute, University of Chicago, Chicago, IL 60637 trsosnic@uchicago.edu freed@uchicago.edu.
authors.forename: Karl F
authors.initials: KF
authors.lastname: Freed
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637; Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637; Computation Institute, University of Chicago, Chicago, IL 60637 trsosnic@uchicago.edu freed@uchicago.edu.
authors.forename: Tobin R
authors.initials: TR
authors.lastname: Sosnick
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D003198:Computer Simulation; D006859:Hydrogen / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D013816:Thermodynamics; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D000596:Amino Acids; D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D006859:Hydrogen
keywords: cooperative folding; folding pathway; hydrogen exchange; protein folding; λ repressor