Amide backbone and water-related H/D isotope effects on the dynamics of a protein folding reaction.
The denaturant-dependent relaxation kinetics of folding and unfolding of an isolated all-beta domain of rat CD2 have been measured at 25 degrees C in four isotopic conditions: with a protonated (amide) backbone in H2O and in D2O and with a deuterated backbone in H2O and in D2O. The data show that this structure, which contains no disulfide bonds, folds through a rapidly formed intermediate (pathway U-I-F) and that all free energy changes between states are insensitive to isotopic substitution of the amide groups required for intrachain hydrogen bonding. However, the folding reaction is significantly influenced by the nature of the bulk solvent. In D2O, the stability of each state in the folding pathway, relative to the unfolded molecule, is enhanced to a degree which is proportional to its m value, a measure of the exposure of nonpolar protein groups to the solvent. Together these observations suggest that, at this temperature, the solvent isotope effect arises from enhanced hydrophobic interactions which, in turn, results from an increased strength of the solvent-solvent hydrogen bond in D2O. Apart from emphasizing the role of bonds between solvent molecules in protein folding, the results also have practical implications for amide H/D exchange studies. While the replacement of amide protons by deuterons will not affect the protein's stability during exchange experiments, it is important to account for the isotopic influence of the solvent in which the exchange reaction is performed.
Parker
M J
MJ
Department of Biochemistry, University of Bristol, School of Medical Sciences, U.K.
Clarke
A R
AR
D000577:Amides / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D018801:CD2 Antigens / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D051381:Rats; D012995:Solubility; D014867:Water / Q000737:chemistry* / Q000378:metabolism
D000577:Amides; D018801:CD2 Antigens; D006146:Guanidines; D014867:Water; D003903:Deuterium; D019791:Guanidine