(application abstract): The chaperonin TRiC is a protein folding nanomachine necessary for the growth of all eukaryotic cells. The protein substrates of this barrel-shaped 16-subunit nanomachine include actins, tubulins, and tumor suppressor proteins. Similarly to its archaeal homolog Mm-cpn, it couples ATP hydrolysis to internalization, folding, and release of newly synthesized polypeptide chains. The folding cycle includes opening and closing of a built-in lid of the chaperonin critical for binding and release. Our Nanomedicine Development Center (NDC) will extend and integrate the current techniques in electron cryomicroscopy, single-molecule imaging, computational biology, and X-ray crystallography to quantify the chaperonin subunit conformations and dynamics as well as the protein folding intermediates bound within the chaperonin cavity. The marriage of these advanced technologies will allow us to visualize chaperonin machinery functioning not only in vitro but also within cells. Building on a more comprehensive and quantitative description of these protein folding nanomachines, we will engineer modified chaperonins to provide a novel therapeutic tool for inhibiting and promoting the folding of selected proteins whose misfolding or aggregation are associated with human diseases. These proteins include actin, tumor suppressor proteins p53 and Von Hippel Lindau, the aggregating A-beta peptide and the cataract related lens protein, gamma-crystallin. Through visualizing conformations and sites in which these chains are folded by the chaperonin, together with their experimentally observed and computed dynamics, we will also design novel substrates that will be folded efficiently in the naturally occurring or newly engineered chaperonin, opening new avenues in protein design. The corresponding approaches will include design of adaptor peptides for modifying the substrates or chaperonins to enhance or inhibit substrate-chaperone interactions. We have assembled a team of 15 investigators from 6 institutions with expertise in chaperones, protein folding, electron cryomicroscopy, computer simulation and modeling, X-ray crystallography, singlemolecule imaging and trapping, and clinical research. We will work together in developing a strategic set of experimental and computational tools that will enable characterization of biological nanomachines, both in vitro and in vivo. We will interact with complementary expertise of other NDC in synthetic chemistry and fluorescence technologies. Our Center can be a critical resource to other NDC who need assistance in solving protein folding and aggregation problems. Our clinical investigators will contribute to the design of pilot studies for therapeutic applications in cell culture models of disease states. We also plan new educational tools via virtual courses in design and application of biological nanomachines, aimed at bridging the gap between biology and mechanical engineering for students in our 6 participating institutions. Finally, the organization, collaborations and communications in our NDC exemplify the 21st century goal of conducting interdisciplinary research via new mechanisms of data sharing and analysis.
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