Biological nanomachines carry out many processes in which mechanical forces are used to trigger conformational transitions. AFM, in its SMFS mode, combined with nanoscale physics, computational techniques, and protein engineering, has established a new field of investigation (i.e., protein mechanics), whose aim is to directly study the mechanical properties of intramolecular (folding/unfolding) and intermolecular (binding/unbinding) protein interactions.

Single-molecule mechanical studies have already produced many remarkable new insights into protein folding and function although only a few protein structures have been investigated so far and the available data are both incomplete and heterogeneous owing to the lack of a standard for the experimental conditions. Also, most of these studies have been necessarily phenomenological, i.e., limited to the characterization of some of the main mechanical properties, but lacking a detailed mechanistic description of the process.

tte first findings have revealed that proteins show a broad range of responses to mechanical stress. It has been found that mechanical proteins tend to be mechanically more stable than nonmechanical ones, ^-strand proteins are usually mechanically more stable than a-helical ones, and ^-strand proteins with a shear mechanical topology were found to be mechanically more stable than zipper ^-strand proteins. Furthermore, patches of hydrogen bonds in the protein backbone have often been identified as the breakpoint of mechanical resistance.

tte identification of the force-bearing components of proteins has begun to yield information regarding the molecular determinants underlying their mechanical behavior. To date, SMFS experiments have demonstrated that the mechanical stability of a protein is not correlated with its thermodynamic stability and that it can be modulated by ligands. It has also been revealed that chemical and mechanical unfolding follow different pathways and have different unfolding barriers. Moreover, it has been established that the mechanical stability and the mechanical unfolding pathway depend on the pulling geometry and the application points of the force. Although the molecular basis of the mechanical resistance of proteins remains in most cases unclear, several determinants have been identified so far: amino acid sequence, topology, unloaded unfolding rate constant, and pulling geometry.

In less than a decade since the first SMFS experiments were carried out on proteins, this technique has identified a variety of components involved in a number of different mechanical functions in the protein machinery. Elastic proteins and elastic protein components (elastin, titin PEVK, and N2B) seem to function mainly as pure entropic springs (with entropic elasticity). In contrast, mechanical modular proteins (like fibronectin, tenascin, or titin Ig domains) seem to rely on module unfolding as a "mechanical buffer" (enthalpic elasticity), in this way behaving as perfect mechanical shock absorbers, ttus, the mostly modular cell adhesion proteins have their elastic elements (modules) connected in series through intermolecular bonds (Fig. 8.2b). When these proteins are subjected to mechanical tension, often present at the cell-cell interface, it has been proposed that this organization helps to maintain the interactions of these adhesive molecules over larger extensions, and increases their lifetime when compared with that of a rigid element (Oberhauser et al. 1998; Evans and Ritchie 1999).

Single-molecule mechanical techniques are still in their infancy, but once this first generation of techniques evolves, we can expect them to provide us with more and more fundamental information on the structure and function of proteins, and to become an indispensable tool in understanding how these molecules fold and work. With the newfound capacity to manipulate and look at the "secret life" of single molecules, we should be prepared for many surprises from the mechanochemis-try of proteins. Indeed, we are entering a new and exciting time in biology which, in combination with the knowledge generated in this proteomic era, is likely to help us understand how proteins work in real time.

Acknowledgements and Notes added in Proof. We are in debt to J. Clarke, H. Li, F. Ritort, and S. García-Manyes for their critical reading of the manuscript. We thank J.M. Fernandez, J. Clarke, and F. Ritort for kindly sending preprints of their current works. We also thank the members of the M.C.-V. laboratory for helpful discussions and comments on the manuscript. Ms work was supported by grants from the former Spanish Ministry of Science and Technology (BI02003-08004), the Consejería de Educación of the Madrid Community (GR/SAL/0836/2004) and the institutional RED CIEN (G03/06) to M.C.-V. and from the National Institutes of Health (DK067443) and the John Sealy Memorial Endowment Fund for Biomedical Research to A.F.O. We apologize to many of our colleagues for omitting or only indirectly citing their work. Owing to the limitations of space, some important contributions to the main points of our review have not been cited and some review articles, rather than the primary sources, have sometimes been preferred as references. While this chapter was in proof several papers have been published reporting important facts for this field, ttus the surprising Hookean behaviour of ankyrin (Lee et al. 2006) as well as the mechanical refolding of ankyrin repeats (Lee et al. 2006) and RNase H (Cecconi et al. 2005); the later study has been done with optical tweezers and found RNase H to be a three-state folding protein; also a recent publication has reported a higher mechanical stability for DHFR (around 130 pN), although at a higher loading rate than previously used (Wilcox et al. 2005); this study has also brought additional evidence supporting the dependence of mitochondrial unfolding on the local structure adjacent to the targetting sequence, similar to the findings from AFM unfolding. Finally, another study suggests that the mechanism for mitochondrial unfolding maybe somehow different from those involved in AFM and chemical techniques (Sato et al. 2005).

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