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D/H amide kinetic isotope effects reveal when hydrogen bonds form during protein folding

Abstract

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 ~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 ~50% of the native H-bonds are formed in the transition state of these helical proteins. In contrast, an α/β 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 ~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 α-helical proteins forming a number of H-bonds proportional to the amount of surface buried in the transition state.

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Figure 1: D/H amide isotope effects.
Figure 2: Change in stability upon backbone amide D/H exchange.
Figure 3: Effects of isotope composition on protein stability.
Figure 4: Effects of amide composition on folding kinetics.
Figure 5: Kinetic isotope effects, m values and protein topologies.

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Acknowledgements

We thank S.W. Englander, N. Kallenbach, K. Shi, T. Pan and X. Fang for numerous enlightening discussions, and B. Golden for useful comments on the manuscript. A ubiquitin expression vector was generously provided by K.D. Wilkinson. We also thank G. Reddy for peptide synthesis. This work was supported in part by a grant from the NIH (T.R.S.) and one from the National Cancer Institute to the University of Chicago Cancer Research Center.

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Correspondence to Tobin R. Sosnick.

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Krantz, B., Moran, L., Kentsis, A. et al. D/H amide kinetic isotope effects reveal when hydrogen bonds form during protein folding. Nat Struct Mol Biol 7, 62–71 (2000). https://doi.org/10.1038/71265

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