title: Measuring hydrogen exchange rates in invisible protein excited states.
abstract: Hydrogen exchange rates have become a valuable probe for studying the relationship between dynamics and structure and for dissecting the mechanism by which proteins fold to their native conformation. Typically measured rates correspond to averages over all protein states from which hydrogen exchange can occur. Here we describe a new NMR experiment based on chemical exchange saturation transfer that provides an avenue for obtaining uncontaminated, per-residue amide hydrogen exchange rates for interconverting native and invisible states so long as they can be separated on the basis of distinct (15)N chemical shifts. The approach is applied to the folding reaction of the Fyn SH3 domain that exchanges between a highly populated, NMR-visible native state and a conformationally excited, NMR-invisible state, corresponding to the unfolded ensemble. Excellent agreement between experimentally derived hydrogen exchange rates of the excited state at a pair of pHs is obtained, taking into account the expected dependence of exchange on pH. Extracted rates for the unfolded ensemble have been used to test hydrogen exchange predictions based on the primary protein sequence that are used in many analyses of solvent exchange rates, with a Pearson correlation coefficient of 0.84 obtained.
authors.affiliation: Departments of Molecular Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; and.
authors.forename: Dong
authors.initials: D
authors.lastname: Long
authors.affiliation: Departments of Molecular Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; and.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bouvignies
authors.affiliation: Departments of Molecular Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; andProgram in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8 kay@pound.med.utoronto.ca.
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D000818:Animals; D002645:Chickens; D006859:Hydrogen / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009584:Nitrogen / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry*; D012984:Software; D012997:Solvents / Q000737:chemistry; D018909:src Homology Domains
chemical_list: D011506:Proteins; D012997:Solvents; D006859:Hydrogen; D051076:Proto-Oncogene Proteins c-fyn; D009584:Nitrogen
keywords: amide exchange with solvent; conformationally excited protein states; protein folding