Zev D. Bryant, Ph.D. - US grants

Engineering Molecular Motors Molecular motors lie at the heart of biological processes from DNA replication to cell migration. The principal goal of my research is to understand the physical mechanisms by which these nanoscale machines convert chemical energy into mechanical work. I propose a radical change in the way my laboratory approaches this goal. We will rigorously challenge our understanding of the relationships between molecular structures and mechanical functions by rationally engineering molecular motors with novel properties. The performance of our designs will illuminate both the inner workings of natural biological motors and the general operational constraints for producing directed motion on the molecular scale. Ultimately, construction of molecular motors to arbitrary specifications will provide a powerful toolkit for synthetic biology, therapeutics, and nanotechnology. My laboratory will design and characterize molecular motor variants using a rapid testing cycle that relies on new instrumentation for high throughput single molecule tracking and manipulation assays. We will focus our efforts by choosing a small number of ambitious design targets, each requiring several intermediate molecular innovations and optimizations. For the period of this award, two initial design targets will be pursued, leveraging our existing expertise in myosin and topoisomerase mechanochemistry: 1) We will create a fast, processive myosin motor that can be optically or chemically signaled to reversibly switch its direction of hand-over-hand movement along an actin filament. 2) We will create a robust tension-insensitive rotary DNA-associated motor that introduces torque in the opposite direction from DNA gyrase. Success will represent an unprecedented level of control over nanoscale motion, building an engineering capacity that will eventually be used to design protein nanoassemblies capable of sophisticated intracellular therapeutic functions such as genome repair. Novel molecular motors will also have ex vivo applications including molecular sorting and assembly of nanoelectronics in microfabricated devices.

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. SUMMARY Biological molecular motors carry out mechanical functions by coupling conformational changes to substeps in chemical fuel consumption. Gyrase is an essential motor that harnesses the free energy of ATP hydrolysis to introduce negative supercoils into DNA. Here we propose a comprehensive biophysical approach for studying gyrase dynamics and mechanochemical coupling. Our goal is to define a set of functionally relevant structural intermediates of the gyrase nucleoprotein complex, and characterize the mechanics and chemistry of transitions between these intermediates. In recent work, we have measured structural dynamics using a single molecule assay in which gyrase drives the processive, stepwise rotation of a submicron fluorescent sphere attached to the side of a stretched DNA molecule. Analysis of rotational pauses and simultaneous measurements of DNA contraction revealed multiple ATP-modulated structural transitions. We showed that a critical DNA wrapping step is coordinated with the ATPase cycle and proceeds via an unanticipated structural intermediate that dominates the kinetics of supercoiling. We proposed a mechanochemical model that quantitatively explained our findings, featuring a conformational landscape of loosely coupled transitions funneling the motor toward productive energy transduction. Our model provides a new framework that guides the investigations in this proposal. In the proposed work, we will exploit new instrumentation and methods that we have recently developed. We can now measure twist in individual stretched DNA molecules with spatiotemporal resolutions that far exceed any previously reported methods, using gold nanospheres as low-drag rotational probes. This technology provides access to millisecond-resolution dynamics of DNA gyrase. We further propose to complement our DNA-centric measurements of nucleoprotein dynamics (relying on observations of changes in twist and extension) with simultaneous measurements of protein domain movements using single molecule fluorescence. Finally, we will use complementary structural approaches to characterize the architectures of substates identified in our single molecule studies. The methods established in this proposal are expected to be an important contribution in themselves, and should be directly applicable to observing the structural dynamics of diverse nucleoprotein complexes ranging from nucleosomes to preinitation complexes.

SUMMARY Diverse cytoskeletal motors perform essential cellular functions including spindle assembly, nuclear positioning, and polarized transport of mRNA, proteins, and membranous cargos along microtubules and actin filaments. Engineering biomolecular motors with tunable and dynamically controllable properties can provide (1) rigorous tests of models relating molecular structures to mechanical functions, (2) novel tools for selective perturbation of mechanical processes inside living cells, and (3) optimized components for complex tasks such as molecular sorting and directed assembly in vitro. This project seeks to develop and characterize a comprehensive set of modified cytoskeletal motors with defined properties ? including speed, direction, and force generation ? than can be controlled using external cues such as light. A modular protein engineering approach will be applied to both actin-based and microtubule-based transport. During successive design cycles, chimeric motors will be constructed based on structural models, and then functionally characterized using gliding filament assays, single fluorophore imaging, gold nanoparticle tracking, and optical trapping. Complementary structural characterization using cryoelectron microscopy will be used to compare the experimental conformations of filament-bound motors to the original structural designs, and to yield new insights into class-specific structure-function relationships. Finally, pilot studies will be conducted to test the function of engineered motors inside living cells. The specific aims of this project are (1) to create diverse myosin motors that exploit dynamic changes in lever arm structure in order to shift gears ? speed up, slow down, or change directions ? when exposed to blue light; (2) to develop diverse microtubule-based motors with artificial lever arms, including light-activated gearshifts, by exploiting a mechanistic analogy between myosins and class-14 kinesins, and (3) to create processive multimeric assemblies of controllable engineered myosins and kinesins, and characterize their force-generating properties. If successful, this work will dramatically expand the potential applications of engineered molecular motors, and provide unprecedented control over nanoscale motion. Genetically encoded light- responsive motors will expand the optogenetics toolkit, complementing precise perturbations of ion channels and intracellular signaling with spatiotemporal control of cytoskeletal transport and contractility. Optogenetic control of bidirectional transport will enable dynamic relocalization of biomolecules and organelles; highly processive and controllable motors will have potential applications in gene and drug delivery; and controllable motors may be used to sort, shuttle, and concentrate analytes in microfabricated diagnostic devices.

SUMMARY Central functions in DNA biology and biotechnology are carried out by nucleoprotein machines. In these dynamic macromolecular assemblies, the DNA duplex is bound and distorted in complex with protein and sometimes RNA. Biophysical measurements and models are needed to understand the mechanisms of these machines, in which coordinated conformational changes in protein and nucleic acid components are coupled with chemical steps such as backbone cleavage or nucleotide hydrolysis. This is a renewal application for a grant in which we previously developed high-resolution and multimodal single-molecule approaches and applied them to elucidate mechanochemical coupling in the ATP-dependent supercoiling motor DNA gyrase from E. coli. Here, we propose to leverage our methods and insights to dissect the dynamics and mechanics of additional nucleoprotein machines, focusing on the RNA-guided nucleases Cas9 and Cas12a and comparing DNA gyrase motors across species. We will characterize substeps in DNA interrogation and DNA supercoiling, molecular determinants of energy landscapes and kinetics, and the effects of mechanical strains experienced in the genome. If successful, the project will determine the physical mechanisms of DNA interrogation by RNA-guided nucleases in dynamic and mechanical detail, providing a quantitative description of the target search process for enzymes that are currently being exploited for gene editing and for a rapidly expanding set of other applications involving specific targeting of activities to sites in the genome. New DNA gyrase measurements will further elucidate biophysical specializations, structural properties, and mechanical regulation of enzymes that are important targets for antibacterial drugs. Finally, single-molecule methods development driven by these biophysical questions will have broad applications in systems ranging from transcription to nucleosome remodeling.