The molecular mechanisms determining the onset of protein aggregation revealed by single molecule force-clamp spectroscopy

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, Mad Cow, eye's cataracts and many others. These diseases have their origin when a single molecule undergoes a conformational change and is not able to fold back into its native structure anymore. This is one of the origins of toxicity; once a protein is unfolded and cannot get back to its folded state, it sticks to a neighboring unfolded protein in a rather fast way. This creates a nucleation seed that eventually results in the presence of aggregates, called amyloids, which are the signature of several diseases. For example, plaques of aggregates are found in the brains of Alzhemier's and Parkinson's disease patients. Perhaps the most conspicuous amyloid disease is cataract formation in the human eye, where the amyloids can be observed directly by looking into an affected eye. Unfortunately, once these amyloid aggregates are detected, it is often too late to act. It is therefore really challenging to discover why and when this aggregative process starts. This project aims to develop a new strategy to able to experimentally manipulate the state of a single protein and probe the time evolution of the different shapes and conformations adopted by each protein during its folding trajectory. The challenge is now to detect the mechanisms why proteins fail to fold, at the level of a single molecule. There are very few and only recent studies of aggregating proteins at the single molecule level. I will use a novel technique, named single molecule force-clamp spectroscopy, 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 evolutionary designed to fold. I will first further the investigations to completely understand the different trajectories followed by an individual protein until it folds. I will then expand this methodology to study the folding behaviour of proteins that cause a great variety of diseases such as Alzheimer's (the Abeta42 polypeptide), Parkinson's (caused by the aggregation of the alpha-synuclein protein) and also the eye's cataract, which is triggered by the misfolding of the protein gammaD crystallin. In all these cases, I will compare the behaviour of these amyloid-forming proteins with those proteins that succeed to fold. I will identify the conformation (folded, unfolded or intermediate) where each of these amyloid-forming proteins departs from the 'functional' folding route. I will finally study if the presence of other companion proteins, called chaperones, assists in the folding mechanism of the aggregating proteins. 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 aggregation, originating at the single molecule level. I seize on the remarkable opportunity of expanding the current applications of force-clamp to solving the common riddle of these diseases. These basic biophysical studies hold great promise for impacting several fields of research, such as the molecular understanding of such devastating human diseases for which there is, at the present time, no cure.

Current studies on protein aggregation have mainly focused on the mechanisms of amyloid formation. However, an emerging consensus is that conformational diseases originate at the single molecule level, when an innocuous monomer undergoes a structural transition into a toxic conformation. To bridge this gap, I will use single molecule force-clamp spectroscopy technique to elucidate the dynamics of single amyloidgenic peptides as they collapse into rare toxic conformations. As a benchmark, I will first study the conformational folding dynamics of monomer proteins evolutionary designed to fold, such as Ubi or I27. I will focus on the characterization of the newly discovered ensemble of collapsed states that hold the key to explaining how an extended polypeptide folds. I will then expand this methodology to study the collapsed conformations of the amyloid forming proteins alpha-synuclein, which causes Parkinson's disease, Abeta42, responsible for Alzheimer's disease, and gammaD-crystallin, which triggers the cataracts in the eye lens. In this vein, I will apply the force-quench protocol to track the kinetics of conformational change of alpha-synuclein, which will be fingerprinted by a different degree of protein extensibility. I will then study the threshold number of contiguous Abeta42 monomers required to trigger protein aggregation. I will complement these studies with SMD simulations, which will provide atomistic insight into the key interactions determining the onset of aggregation. Finally, I will uncover the (un)folding dynamics of the gammaD-crystallin protein. I will track the folding mechanism for those proteins exhibiting hereditary mutations. Finally, I will study the mechanisms by which chaperones inhibit aggregation. The uncanny ability of single molecule techniques to observe the acquisition of rare misfolded conformations will help establish mechanistic paradigms for developing a unified molecular scale understanding of the origins of these diseases.

Impact on non-academic research. I will develop new strategies to probe the initial stages of protein misfolding and aggregation, leading to many devastating diseases that unfortunately are having a growing impact on the society. The capability of testing new therapeutic strategies (such as new drugs) at the single molecule level will potentially have a positive impact on UK industry. Conversely, our proposed single molecule experiments could provide a first step into the design of new drugs able to block particular conformations that can be now singled out using our single molecule approach. I will use the local expertise of King's College Business Ltd. On Knowledge Transfer, Intellectual Property, Consultancies and Commercial Partnerships to facilitate interactions with the commercial sector.
Impact on tools and technology. The proposed research will create new experimental tools. These would include new expertise in force protocols for optimal capture of the different conformations visited by a single protein along its individual folding pathway. It is plausible that only one of these conformations is effectively reactant to particular drugs, and that this particular species could direct the aggregation process. Acting selectively on this particular conformation would have a big impact on the further development of the disease. Furthermore, I will improve the temporal resolution of our single molecule spectrometer (with e.g. faster piezo-electric designs) to eventually capture key intermediate species that are too transient to be captured with the available bandwidth.
Impact on Health. An obvious benefit of our research proposal entails the basic knowledge regarding the initial stages of aggregative or conformational diseases. Any new discovery regarding the initial stages of aggregation, occurring at the single molecule level, might have a huge impact on the treatment of the particular disease. I will exploit intense collaborations with different research groups working on the aggregative diseases at different length-scales and also with industries to cooperatively design new strategies to unveil the enigmatic origin of such devastating diseases.
Impact on People. The postdoctoral research assistant will be trained in a multi-disciplinary scientific environment and will learn how to build communication between theoreticians and experimentalists. These skills will improve his employment prospects in both industry and academia.
Impact on UK Competitiveness. The UK's position as a world leader in biomedical and pharmaceutical research is based on innovative ideas and approaches, requiring a range of resources and skills. The project brings together the expertise of people and institutes with the essential skills and resources to develop and test an innovative methodology for capturing the very first stages of protein aggregation, occurring at the single molecule level. This methodology could be used to identify the specific conformations that determine the fate of a particular protein, which could be ideally targeted by specific drugs able to identify them. This experimental approach has the potential to improve our understanding of disease onset and progression. Therefore, this project is likely to increase the UK's scientific and economic competitiveness.
Communication and engagement. The results arising from the proposed research will be published in peer-reviewed journals with high international visibility. During his research career, Garcia-Manyes has demonstrated his ability at publishing his research in the most prominent research journals in his area. The scientific results stemming from this research will also be presented in international meetings and conferences. Of particular importance, Garcia-Manyes has co-organized for the first time in 2010 a workshop in single molecule mechanics in Bilbao, Spain. The plan is now to repeat the workshop once a year in different parts of the world.