Structural Dynamics of Molecular Motors and the Ribosome

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Summary Protein synthesis and active transport of vesicular cargoes are vital to development of all tissues and to the targeted delivery of organelles, proteins, and signaling molecules in eukaryotes. Accordingly, defects in protein expression and transport are linked to developmental, neurodegenerative, pigmentation, immunological, and other diseases. Knowing the detailed mechano-chemistry and structural dynamics of the ribosome and motor proteins is essential for understanding and interpreting their roles in the cell. We have developed a number of powerful new biophysical tools that reveal the structural dynamics and reaction kinetics of the protein synthesis elongation cycle and cargo transport in muscle and non-muscle molecular motors under applied mechanical force. We will apply these unique tools to investigate the rhythm of protein synthesis and premature termination in eukaryotes. We will elucidate the divergent biochemical and mechanical properties of skeletal muscle myosin and non-muscle myosins-I, V, VI and X. Understanding functional dynamics and mechanistic detail that have not yet previously been accessible is now feasible. This MIRA grant coalesced 3 former NIH grants: the applicant's section of a program project on molecular motors in cells, an individual R01 grant to the applicant on basic biophysical mechanisms of molecular motors, and a multi-PI grant on protein synthesis. The links between all of these different topics are that they are subject to formidable study by single molecule biophysics approaches and they incorporate P-loop NTPases with many common structural motifs and principles. They can be under- stood synergistically by studying and comparing their individual structural, energetic and dynamic features. Ex- amples of this synergy are given in the body of the application. For the renewal period we plan to 1) continue the successful development of state-of-the-art single molecule fluorescence and optical trap technology, 2) apply these methods to a series of myosin isoforms that have been described in the literature as having qualitatively different properties, 3) build a new class of intracellular force-FRET sensors for studying mechanobiological signaling from the peripheral environment of a cell to control of gene expression in the nucleus, 4) compare and contrast mechanisms of eukaryotic protein synthesis with the bacterial system, 5) elucidate the detailed mecha- nisms for enhancement, during protein synthesis, of premature termination codon (PTC) read-through by phar- maceuticals that are candidates for therapy in PTC diseases (e.g. Duchenne muscular dystrophy and cystic fibrosis,) and 5) a new venture to test processive translocation by AAA+ domain ring proteins, including Hsp104 (which disaggregates toxic amyloid proteins) and katanin (which modulates microtubule length by severing and is also tied to diseases). Overall, these studies will lead to a much more general view of the mechanisms and characteristics of the ribosome and molecular motors in vitro and in live cells leading to a more rigorous under- standing of their functions in cell biology and disease.