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Conformational strain in the hydrophobic core and its implications for protein folding and design

Nature Structural Biology volume 9, pages 485–493 (2002)Cite this article

Abstract

We have designed de novo 13 divergent spectrin SH3 core sequences to determine their folding properties. Kinetic analysis of the variants with stability similar to that of the wild type protein shows accelerated unfolding and refolding rates compatible with a preferential stabilization of the transition state. This is most likely caused by conformational strain in the native state, as deletion of a methyl group (Ile→Val) leads to deceleration in unfolding and increased stability (up to 2 kcal mol−1). Several of these Ile→Val mutants have negative φ‡−U values, indicating that some noncanonical φ‡−U values might result from conformational strain. Thus, producing a stable protein does not necessarily mean that the design process has been entirely successful. Strained interactions could have been introduced, and a reduction in the buried volume could result in a large increase in stability and a reduction in unfolding rates.

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Figure 1: Ribbon diagram of the spectrin SH3 domain.
Figure 2: Rectangular cladograms for the hydrophobic core of 63 selected SH3 sequences and the designed hydrophobic core mutants.
Figure 3: Urea denaturation profiles of the WT and the 13 core mutants.
Figure 4: Folding and unfolding kinetic curves of the WT, spectrin SH3 and core mutants.
Figure 5: Stereo view of the omit map of the core residues.
Figure 6: Superimposition of the backbones corresponding to the WT and different core mutants.
Figure 7: Urea denaturation profiles of the WT-spectrin SH3 and core mutants.

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Acknowledgements

C. Vega was supported by a Marie Curie fellowship. This work was partly supported by an EU Biotech grant. We are very grateful to M. DelaPaz for her critical comments.

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  1. Salvador Ventura and Maria Cristina Vega: These authors have contributed equally to this work.

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  1. European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, D-69117, Germany

    Salvador Ventura, Maria Cristina Vega, Emmanuel Lacroix, Isabelle Angrand, Laura Spagnolo & Luis Serrano

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Correspondence to Luis Serrano.

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Ventura, S., Cristina Vega, M., Lacroix, E. et al. Conformational strain in the hydrophobic core and its implications for protein folding and design. Nat Struct Mol Biol 9, 485–493 (2002). https://doi.org/10.1038/nsb799

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