Making and measuring macromolecular machines

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Synopsis

Molecular machines lie at the heart of biological processes ranging from DNA replication to cell migration. We use single-molecule tracking and manipulation to characterize the structural dynamics of these nanoscale assemblies, and further challenge our understanding by designing and testing structural variants with novel properties that expand the functional range of known biomolecular machines. In the process, we are developing an engineering capacity for molecular motors with tunable and dynamically controllable physical properties, providing a toolkit for precise perturbations of mechanical functions. We have previously developed a family of light-responsive myosin motors, enabling precise control of fast and processive molecular transport in vitro and in living cells. I will describe our ongoing efforts to augment and diversify engineered cytoskeletal motors, including newly developed light-responsive filamentous myosins for control of contractility. I will further discuss our measurements of dynamics and mechanics in CRISPR endonucleases. In the latter work, we have used high-resolution multimodal single-molecule methods to study the process of DNA interrogation by Cas9 and Cas12a.  We have observed intermediate steps in target recognition and probed important effects of DNA torsion on the dynamics and specificity of these nucleoprotein machines.