Mapping the topography of a protein energy landscape

Protein energy landscapes are highly complex, yet the vast majority of states within them tend to be invisible to experimentalists. Here, using site-directed mutagenesis and exploiting the simplicity of tandem-repeat protein structures, we delineate a network of these states and the routes between them. We show that our target, gankyrin, a 226-residue 7-ankyrin-repeat protein, can access two alternative (un)folding pathways. We resolve intermediates as well as transition states, constituting a comprehensive series of snapshots that map early and late stages of the two pathways and show both to be polarized such that the repeat array progressively unravels from one end of the molecule or the other. Strikingly, we find that the protein folds via one pathway but unfolds via a different one. The origins of this behavior can be rationalized using the numerical results of a simple statistical mechanics model that allows us to visualize the equilibrium behavior as well as single-molecule folding/unfolding trajectories, thereby filling in the gaps that are not accessible to direct experimental observation. Our study highlights the complexity of repeat-protein folding arising from their symmetrical structures; at the same time, however, this structural simplicity enables us to dissect the complexity and thereby map the precise topography of the energy landscape in full breadth and remarkable detail. That we can recapitulate the key features of the folding mechanism by computational analysis of the native structure alone will help toward the ultimate goal of designed amino-acid sequences with made-to-measure folding mechanisms—the Holy Grail of protein folding

Context-dependent energetics of loop extensions in a family of tandem-repeat proteins

Consensus-designed tetratricopeptide repeat proteins are highly stable, modular proteins that are strikingly amenable to rational engineering. They therefore have tremendous potential as building blocks for biomaterials and biomedicine. Here, we explore the possibility of extending the loops between repeats to enable further diversification, and we investigate how this modification affects stability and folding cooperativity. We find that extending a single loop by up to 25 residues does not disrupt the overall protein structure, but, strikingly, the effect on stability is highly context-dependent: in a two-repeat array, destabilization is relatively small and can be accounted for purely in entropic terms, whereas extending a loop in the middle of a large array is much more costly because of weakening of the interaction between the repeats. Our findings provide important and, to our knowledge, new insights that increase our understanding of the structure, folding, and function of natural repeat proteins and the design of artificial repeat proteins in biotechnology

Spectroscopic studies of protein hydration and folding

SIGLEAvailable from British Library Document Supply Centre- DSC:D063053 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

From artificial antibodies to nanosprings: the biophysical properties of repeat proteins

In this chapter we review recent studies of repeat proteins, a class of proteins consisting of tandem arrays of small structural motifs that stack approximately linearly to produce elongated structures. We discuss the observation that, despite lacking the long-range tertiary interactions that are thought to be the hallmark of globular protein stability, repeat proteins can be as stable and as coorperatively folded as their globular counterparts. The symmetry inherent in the structures of repeat arrays, however, means there can be many partly folded species (whether it be intermediates or transition states) that have similar stabilities. Consequently they do have distinct folding properties compared with globular proteins and these are manifest in their behaviour both at equilibrium and under kinetic conditions. Thus, when studying repeat proteins one appears to be probing a moving target: a relatively small perturbation, by mutation for example, can result in a shift to a different intermediate or transition state. The growing literature on these proteins illustrates how their modular architecture can be adapted to a remarkable array of biological and physical roles, both in vivo and in vitro. Further, their simple architecture makes them uniquely amenable to redesign—of their stability, folding and function—promising exciting possibilities for future research

Functionalised staple linkages for modulating the cellular activity of stapled peptides

10.1039/c4sc00045eChemical Science551804-1809CSHC

Predicting experimental quantities in protein folding kinetics using stochastic roadmap simulation

10.1007/11732990_34Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics)3909 LNBI410-42