Protein folding intermediates and pathways studied by hydrogen exchange.
In order to solve the immensely difficult protein-folding problem, it will be necessary to characterize the barriers that slow folding and the intermediate structures that promote it. Although protein-folding intermediates are not accessible to the usual structural studies, hydrogen exchange (HX) methods have been able to detect and characterize intermediates in both kinetic and equilibrium modes--as transient kinetic folding intermediates on a subsecond time scale, as labile equilibrium molten globule intermediates under destabilizing conditions, and as infinitesimally populated intermediates in the high free-energy folding landscape under native conditions. Available results consistently indicate that protein-folding landscapes are dominated by a small number of discrete, metastable, native-like partially unfolded forms (PUFs). The PUFs appear to be produced, one from another, by the unfolding and refolding of the protein's intrinsically cooperative secondary structural elements, which can spontaneously create stepwise unfolding and refolding pathways. Kinetic experiments identify three kinds of barrier processes: (a) an initial intrinsic search-nucleation-collapse process that prepares the chain for intermediate formation by pinning it into a condensed coarsely native-like topology; (b) smaller search-dependent barriers that put the secondary structural units into place; and (c) optional error-dependent misfold-reorganization barriers that can cause slow folding, intermediate accumulation, and folding heterogeneity. These conclusions provide a coherent explanation for the grossly disparate folding behavior of different globular proteins in terms of distinct folding pathways.
Johnson Research Foundation, Philadelphia, Pennsylvania, USA. walter@HX2.Med.upenn.Edu
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D003574:Cytochrome c Group / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D013997:Time Factors
D003574:Cytochrome c Group; D006859:Hydrogen; D019791:Guanidine