The Fold Approach

Background:

Despite the importance of recognising complexity, fundamental investigations of the folding of small proteins remain at the heart of protein folding studies. Certain well-understood model systems have been central in the development of new conceptual frameworks. They provide test-beds for new theories and methods. Our group was the first to systematically set out to study the folding of families of related proteins - and now this has become an important tool in many laboratories¹.

The contrasting topologies of some of the folds being studied by our group.

Mechanisms and pathway:

Studies of the folding of related proteins suggest that topology, the nature of the secondary structure content and sequence variations all play a role in determining how a protein folds². A recent theoretical study suggests that topology is likely to be the dominant factor in determining folding pathways in all-beta proteins where long-range interactions predominate³. However, in all-alpha proteins, dominated by local interactions, sequence changes are more likely to result in variable folding pathways within a family.

Oliveberg and co-workers have analysed the nucleation of folding in a number of different families⁴. They describe nucleation motifs as "foldons", and suggest that they correspond to the smallest elements with sufficient stabilising interactions to overcome their loss in chain entropy⁵. The nuclei of the topologically complex Greek-key proteins comprise four elements of secondary structure⁶. Our investigations of basic folding studies aim to address the relative importance of topological complexity, chain connectivity, and secondary structure content on the folding mechanisms of proteins.

Landscape roughness - general or rare?

One of the principal observations from energy landscape theory is that, in general, evolution selects for smooth, frictionless landscapes, and that frustration (internal friction) would slow folding⁷. Our work, published in Nature in 2011, was the first direct demonstration of both the existence and relevance of significant landscape roughness in naturally occurring, slow-folding proteins (spectrin domains)⁸. This friction was observed by analysis of the viscosity dependence of the folding kinetics⁹. We proposed that introducing landscape roughness might be a mechanism whereby a protein might be able to be "long-lived" in vivo. We now intend to investigate the phenomenon of landscape roughness in more depth.

We wish to:

• understand the mechanism by which roughness is conferred.
• ask whether this is a common phenomenon that has not been detected because it has not been looked for.
• look for evolutionary evidence that roughness in proteins might be conserved as a protective mechanism against unfolding.

Current projects

• Spectrin Domains: investigating why some spectrin domains (e.g. R15) have low internal friction and fold rapidly, whereas almost identical sequences (e.g. R16 and R17) fold far more slowly with substantial internal friction.
• Death Domains: investigating whether secondary structure, chain connectivity or overall topology is most important for protein folding and stability.
• MATH domains: investigating whether circular permutants of β-sandwich proteins show altered folding mechanisms and increased susceptibility to aggregation.
• LysM domains: investigating whether small α+β proteins have transition states that are highly robust and resistant to mutation.

References

1. Nickson, A. A. & Clarke, J. (2010). What lessons can be learned from studying the folding of homologous proteins? Methods 52, 38-50.
2. Zarrine-Asfar, A., Larson, S. M. & Davidson, A. R. (2005). The family feud: Do proteins with similar structures fold via the same pathway? Curr. Opin. Struct. Biol. 15, 42-49.
3. Cho, S. S., Levy, Y. & Wolynes, P. G. (2009). Quantitative criteria for native energetic heterogeneity influences in the prediction of protein folding kinetics. Proc. Natl. Acad. Sci. USA 106, 434-439.
4. Shen, T. Y., Hofmann, C. P., Oliveberg, M. & Wolynes, P. G. (2005). Scanning malleable transition state ensembles: Comparing theory and experiment for folding protein U1A. Biochemistry 44, 6433-6439.
5. Lindberg, M. O. & Oliveberg, M. (2007). Malleability of protein folding pathways: a simple reason for complex behaviour. Curr. Op. Struct. Biol. 17, 21-29.
6. Lappalainen, I., Hurley, M. G. & Clarke, J. (2008). Plasticity within the obligatory folding nucleus of an immunoglobulin-like domain. J. Mol. Biol. 375, 547-559.
7. Bryngleson, J. D. & Wolynes, P. G. (1989). Intermediates and barrier crossing in a random energy model (with applications to protein folding). J. Phys. Chem. 93, 6902-6915.
8. Wensley, B. G., Batey, S., Bone, F. A. C., Chan, Z. M., Tumelty, N. R., Steward, A., Kwa, L. G., A., B. & Clarke, J. (2010). Experimental evidence for a frustrated energy landscape in a three-helix bundle protein family. Nature 463, 685-688.
9. Cellmer, T., Henry, E. R., Hofrichter, J. & Eaton, W. A. (2008). Measuring internal friction of an ultrafast-folding protein. Proc. Natl Acad. Sci. USA 105, 18320-18325.