The nanomechanics of a single protein

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.

The gain in knowledge from the proposed research might have impact on the nation's health and wealth in several different ways. First, increase in basic knowledge about fundamental processes occurring in the human body, and understanding how and why diseases occur increased the cultural background of society. Secondly, controlling the outcome of a chemical reaction by applying mechanical force to the reagent system at the industrial scale might turn out to be a tour-de-force in some of the industries that deal with, e.g. particular polymer isomers. This could potentially have an important economic impact. On a deeper layer of knowledge, the increase in our basic knowledge increases the competitiveness of UK universities, thereby allowing them to attract more foreign students. Finally, staff working on the proposed research project will acquire research and professional skills that they might apply later in their careers, either as researchers, academics, or in the private industry field. It is expected that the PDRAs will present their work at numerous international conferences and in results in high impact scientific journals. King's College London is also committed to ensuring that research staff and students develop research, vocational and entrepreneurial skills that are matched to the demands of their future career paths. Both the Department of Physics and the Randall Division are a multidisciplinary environment and as such also provides an excellent place for training researchers at any level in new techniques.
We plan to engage the beneficiaries in several different ways. First, we will present the on-going research in three already scheduled public lectures. The first will take place in the framework of the London Structural Biology meetings organized by UCL. The second will take place in a soiree of the London Thomas Young Centre for Theory and Simulation of Materials, and the third in the specialized and world-reputed Gordon Conference on Single Molecules. Second, we plan to organize a workshop in single molecule mechanics. The PI (Dr. Garcia-Manyes) co-organized in 2010 a workshop in Bilbao, Spain, where students from the whole Europe had the opportunity to perform hands-on experiments and to analyse their own data during an intensive week of work. The plan is now to repeat this workshop in central London. Finally, the Public Relations Department at King's College London regularly publishes press releases reporting the results of its researchers; this information is communicated through the Web and through magazines, newsletters and annual reports for the general public, which are widely distributed. All these media channels will be used as appropriate and feasible to communicate the results of the present project to the public. School pupils attend our Open Days and undertake work experience in our laboratories.
Beneficiaries in the biotechnology and materials science industry will be engaged through King's College London Business Ltd. That provides a getaway to access the wealth of knowledge and expertise available at King's College London. KCL Business has extensive experience to create significant partnerships with industry to maximize innovation and to add value to our partner's business goals and help increase their global effectiveness. KCL Business offers access to knowledge and people, consultancy, collaborative and contract research, the opportunity to develop and licence new intellectual property and to engage with newly created spin-out companies through partnership and investment. If new discoveries will be made during the course of the proposed research, the PI will liaise with KCL Business to assess potential exploitability of the discovery before any publication. KCL Business is well experienced in providing advice in intellectual property rights and will ensure successful protection of intellectual property by patenting novel inventions and discoveries.