The nanomechanics of a single protein

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Each organ in our body is composed of a large number of individual cells working together in a coordinated fashion. Inside each cell, there are thousands of different proteins that perform their function in a very well-established and synchronized way. In general, each of these proteins can be found in two different shapes -the folded and the unfolded states. Proteins unfold and refold continuously in our bodies once they are expressed in the ribosomes, which are the small factories where they are produced. Most proteins are 'active' or 'functional' only when they are in their folded state. Failing to fold gives rise to a myriad of devastating diseases such as Alzhemier's, Parkinson's, BSE (Mad Cow Disease) and many others. Therefore, we need experimental techniques able to track the folding routes of each individual protein undergoing a folding reaction to identify where and why each individual protein deviates from the 'correct' folding highway, being trapped at an intermediate state. This can be now be addressed by using state-of-the-art single molecule force-clamp spectroscopy. Using this approach, proteins are unfolded by the presence of a low (a few piconewtons) mechanical force, and once the force is reduced, the protein folds from highly extended states. Indeed, there are many proteins in our body that are continuously performing their function under the effect of a mechanical force. For example, the proteins involved in muscle elasticity, with crucial function also in e.g. the heart tissue, have to stretch and relax in a reversible way thousands of time every day. Failing to do that might have tragic consequences, resulting in muscle atrophy and, in the most severe cases, cardiac myopathies. Therefore, understanding how a mechanical force controls protein folding in these proteins is of capital importance, and it is far from being understood. In order to control muscle elasticity protein elasticity, nature has devised internal 'locks', called disulfide bonds, which prevent the protein to overstretch under high stress conditions. Such internal mechanical clamps can be mechanically 'open' through a covalent chemical reaction when required. Therefore, understanding the mechanisms to control these 'mechanical switches' is also of paramount importance in biophysics. I will use the novel single molecule force-clamp spectroscopy technique to study the different trajectories followed by an unfolded protein in its journey to the native state. This technique has already proved successful at identifying, for the first time, the different conformations adopted by a protein that has been evolutionarily designed to fold within biological timescales. However, little is known about the mechanisms employed by 'mechanical proteins' to reversibly fold against a pulling force on a short timescale and without the intervention of energy spending mechanisms. I will investigate the conformational dynamics of a series of key proteins that control elasticity in the muscle, in the cytoskeleton and in the extracellular matrix. Next, I will study the effect of force on the reduction of a single disulfide bond embedded within the protein core. In particular, I will study how forces changes the outcome of a chemical reaction, and I will characterize the structure of the 'critical summit point' of the reaction, called transition state, which contains the relevant chemical information on the reaction outcome. Finally, I will examine how disulfide bonds affect the folding of a single protein, a phenomenon occurring in vivo to a wide variety of proteins composing the extracellular matrix. Altogether, these single molecule techniques have now reached a level of maturity where they can be used to attack more significant challenges in biology such as the basic biological mechanisms leading to protein protein and misfolding, especially in these proteins where preserving mechanical extensibility is key to maintain their physiological function.