Proper folding is crucial to achieving a protein?s unique three dimensional structure while the conformational dynamics of the protein play a major role in its biological function. Although significant progress has been made in understanding the folding/unfolding and conformational dynamics for single-domain proteins, these two fundamental processes remain largely unexplored for multi-domain proteins, which have been suggested to account for up to 80% of all eukaryotic proteins. Therefore, a significant disparity exists in our understanding of the underlying mechanisms of folding/unfolding and conformational dynamics for the majority of human proteins and we seek to address these uncertainties through theoretical and experimental investigation. In this proposal, we devise a comprehensive strategy to answer the above, in-depth mechanistic unknowns regarding multi-domain proteins through an energy landscape approach with subsequent experimental validation. The energy landscape approach significantly improves technical capabilities through the establishment of theoretical models for uncovering underlying mechanisms. By establishing the microscopic energy landscape and structure based models, we will elucidate the folding/unfolding mechanisms of DPO4, a multi-domain, model Y-family DNA polymerase critical for bypassing unrepaired DNA lesions, in vitro and in vivo (here means mimicking in vivo conditions), and predict possible intermediate states and critical residues under various environments, including the presence of the ribosome (co-translational) and a crowding agent (in vivo), as well as different thermal and chemical denaturant conditions. Through our microscopic energy landscape and structure based models, we will reveal the underlying mechanisms of conformational changes between various conformational states of DPO4 upon binding to DNA or a protein replication factor PCNA through quantifying the stability, kinetics, and structural hot spots critical for function. The theoretical model predictions will be tested and validated through stopped-flow, circular dichroism, fluorescence energy transfer, and other spectroscopic experiments. The results generated from the proposal will advance the DNA polymerase field while the methods developed here are general and can serve as a framework for studies of folding/unfolding and conformational dynamics of other multi-domain proteins. Moreover, the intricacies of protein folding/unfolding and conformational transitions revealed by our proposed studies will facilitate protein design and drug discovery.
Most macromolecules in any organisms including human beings are proteins. Each protein has a three dimensional structure which is formed from one or more structural lobes named domains. In biology, how a protein folds or unfolds its three dimensional structure and how each domain moves relative to other domains are critical to the protein?s cellular function and interactions with other biomolecules. However, these important fundamental processes have not been clearly understood and will be thoroughly investigated in this proposal through collaboration between a theoretical group and an experimental research laboratory.