A model of the changes in denatured state structure underlying m value effects in staphylococcal nuclease.
Hydrogen exchange kinetics were measured on the native states of wild type staphylococcal nuclease and four mutants with values of mGuHCl (defined as dDeltaG/d[guanidine hydrochloride]) ranging from 0.8 to 1.4 of the wild type value. Residues within the five-strand beta-barrel of wild type and E75A and D77A, two mutants with reduced values of m GuHCl, were significantly more protected from exchange than expected on the basis of global stability as measured by fluorescence. In contrast, mutants V23A and M26G with elevated values of mGuHCl approach a flat profile of more or less constant protection independent of position in the structure. Differences in exchange protection between the C-terminus and the beta-barrel region correlate with mGuHCl, suggesting that a residual barrel-like structure becomes more highly populated in the denatured states of m- mutants and less populated in m+ mutants. Variations in the population of such a molten globule-like structure would account for the large changes in solvent accessible surface area of the denatured state thought to underlie m value effects.
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Department of Biological Chemistry, The Johns Hopkins University of School of Medicine, Baltimore, Maryland 21205, USA.
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D019943:Amino Acid Substitution; D003903:Deuterium / Q000378:metabolism; D017666:Deuterium Oxide / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D019791:Guanidine; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011522:Protons; D012997:Solvents; D013210:Staphylococcus / Q000201:enzymology*; D013816:Thermodynamics
D011522:Protons; D012997:Solvents; D006859:Hydrogen; D003903:Deuterium; D008836:Micrococcal Nuclease; D017666:Deuterium Oxide; D019791:Guanidine