Individual molecules of the giant protein titin span the A-bands and I-bands that make up striated muscle. The I-band region of titin is responsible for passive elasticity in such muscle, and contains tandem arrays of immunoglobulin domains. One such domain (I27) has been investigated extensively, using dynamic force spectroscopy and simulation. However, the relevance of these studies to the behaviour of the protein under physiological conditions was not established. Force studies reveal a lengthening of I27 without complete unfolding, forming a stable intermediate that has been suggested to be an important component of titin elasticity. To develop a more complete picture of the forced unfolding pathway, we use mutant titins--certain mutations allow the role of the partly unfolded intermediate to be investigated in more depth. Here we show that, under physiological forces, the partly unfolded intermediate does not contribute to mechanical strength. We also propose a unified forced unfolding model of all I27 analogues studied, and conclude that I27 can withstand higher forces in muscle than was predicted previously.

Energy landscape theory is a powerful tool for understanding the structure and dynamics of complex molecular systems, in particular biological macromolecules1. The primary sequence of a protein defines its free energy landscape, and thus determines the folding pathway and the rate constants of folding and unfolding, as well as its native structure. Theory has shown that roughness in the energy landscape will lead to slower folding1, but derivation of detailed experimental descriptions of this landscape is challenging. Simple folding models2,3 show that folding is significantly influenced by chain entropy; proteins where the contacts are local fold fast, due to the low entropy cost of forming stabilising, native contacts during folding4,5. For some protein families, stability is also a determinant of folding rate constants6. Where these simple metrics fail to predict folding behaviour it is probable that there are features in the energy landscape that are unusual. Such general observations cannot explain the folding behaviour of the R15, R16 and R17 domains of α-spectrin. R15 folds ~3000 times faster than its homologues, although they have similar structures, stabilities and, as far as can be determined, transition state stabilities7-10. Here we show that landscape roughness (internal friction) is responsible for the slower folding and unfolding of R16 and R17. We use chimeric domains to demonstrate that this internal friction is a property of the core, and suggest that frustration in the landscape of the slow folding spectrin domains may be due to mis-docking of the long helices during folding. Although theoretical studies have suggested that rugged landscapes will result in slower folding, this is the first time that such a phenomenon has been shown experimentally to directly influence the folding kinetics of a “normal” protein with a significant energy barrier – one which folds on a relatively slow ms-s timescale.

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Atomic force microscopy (AFM) experiments have provided intriguing insights into the mechanical unfolding of proteins such as titin I27 from muscle, but will the same be possible for proteins that are not physiologically required to resist force? We report the results of AFM experiments on the forced unfolding of barnase in a chimeric construct with I27. Both modules are independently folded and stable in this construct and have the same thermodynamic and kinetic properties as the isolated proteins. I27 can be identified in the AFM traces based on its previous characterization, and distinct, irregular low-force peaks are observed for barnase. Molecular dynamics simulations of barnase unfolding also show that it unfolds at lower forces than proteins with mechanical function. The unfolding pathway involves the unraveling of the protein from the termini, with much more native-like secondary and tertiary structure being retained in the transition state than is observed in simulations of thermal unfolding or experimentally, using chemical denaturant. Our results suggest that proteins that are not selected for tensile strength may not resist force in the same way as those that are, and that proteins with similar unfolding rates in solution need not have comparable unfolding properties under force.