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Context-dependent contributions of backbone hydrogen bonding to β-sheet folding energetics

Nature volume 430, pages 101–105 (2004)Cite this article

Abstract

Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis1,2,3. Here we have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small β-sheet protein4, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl5,6,7,8,9,10,11,12,13. We perturbed the 11 backbone H-bonds of the PIN WW domain by synthesizing 19 amide-to-ester mutants. Thermodynamic studies on these variants show that the protein is most destabilized when H-bonds that are enveloped by a hydrophobic cluster are perturbed. Kinetic studies indicate that native-like secondary structure forms in one of the protein's loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence. Collectively, our results provide an unusually detailed picture of the folding of a β-sheet protein.

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Figure 1: Structures of the PIN WW domain.
Figure 2: Plot of -ΔΔGf at 2 °C for the amide-to-ester (A-to-E) mutants versus mutation location. ΔΔGf = (ΔGf (wild type) - ΔGf (A-to-E mutant)).
Figure 3: Plot of ΦM versus mutation location.

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Acknowledgements

We thank S. You, J. Blankenship and R. Balambika for discussions. We acknowledge financial support from the NIH, The Skaggs Institute for Chemical Biology, the Lita Annenberg Hazen Foundation and the Norton B. Gilula Fellowship (to S.D.). M.G. and H.N. were supported by a grant from the NSF. P.E.D. was supported by the NIH and the Alfred P. Sloan Foundation.

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Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, BCC 506, La Jolla, California, 92037, USA

    Songpon Deechongkit, Evan T. Powers, Philip E. Dawson & Jeffery W. Kelly

  2. Department of Cell Biology and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, BCC 506, La Jolla, California, 92037, USA

    Philip E. Dawson

  3. Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois, 61801, USA

    Houbi Nguyen & Martin Gruebele

  4. Department of Chemistry, Physics, and Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, Illinois, 61801, USA

    Martin Gruebele

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  1. Songpon Deechongkit
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Correspondence to Jeffery W. Kelly.

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Supplementary information

Supplementary Information

Contains the raw data used to calculate ΦM values (two figures and one table), and a plot of ΦM as a function of temperature for all of the A-to-E mutants with TM > 35 °C. (PDF 187 kb)

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Deechongkit, S., Nguyen, H., Powers, E. et al. Context-dependent contributions of backbone hydrogen bonding to β-sheet folding energetics. Nature 430, 101–105 (2004). https://doi.org/10.1038/nature02611

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