D/H amide kinetic isotope effects reveal when hydrogen bonds form during protein folding.
We have exploited a procedure to identify when hydrogen bonds (H-bonds) form under two-state folding conditions using equilibrium and kinetic deuterium/hydrogen amide isotope effects. Deuteration decreases the stability of equine cytochrome c and the dimeric and crosslinked versions of the GCN4-p1 coiled coil by approximately 0. 5 kcal mol-1. For all three systems, the decrease in equilibrium stability is reflected by a decrease in refolding rates and a near equivalent increase in unfolding rates. This apportionment indicates that approximately 50% of the native H-bonds are formed in the transition state of these helical proteins. In contrast, an alpha/beta protein, mammalian ubiquitin, exhibits a small isotope effect only on unfolding rates, suggesting its folding pathway may be different. These four proteins recapitulate the general trend that approximately 50% of the surface buried in the native state is buried in the transition state, leading to the hypothesis that H-bond formation in the transition state is cooperative, with alpha-helical proteins forming a number of H-bonds proportional to the amount of surface buried in the transition state.
Krantz
B A
BA
Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637, USA.
Moran
L B
LB
Kentsis
A
A
Sosnick
T R
TR
D000577:Amides / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins; D003903:Deuterium / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D013816:Thermodynamics; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000737:chemistry / Q000378:metabolism
D000577:Amides; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D004220:Disulfides; D005656:Fungal Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D014452:Ubiquitins; D014867:Water; D011392:Proline; D003903:Deuterium; D011494:Protein Kinases