Highly Processive DNA Origami Nanoscale Motors

Tuesday, February 18, 2020 enzymatic system consisting of the fiber-conjugated self-activating kinase and an opposing soluble phosphatase. We have previously shown that a soluble mixture of the kinase, called Aurora B, and the inactivating phosphatase generate a bi-stable switch for kinase activation, driven by the ability of the Aurora B kinase to autoactivate in-trans. We hypothesize that compressing the hydrogel with integrated Aurora B kinase will increase its local concentration above threshold, eliciting a switch-like activation and non-linear spatial regimes. As a proof-of-principle approach, we integrated the Aurora B kinase with the collagen-containing hydrogel by conjugating NHS-ester O6-benzylguanine to collagen fibers, followed by covalent linking of the SNAP-tagged Aurora B. Kinase activity within the hydrogels is visualized via a sox-based phosphosensor which increases its fluorescence upon phosphorylation. To study kinase activation within the hydrogel in real time, we constructed a special cuvette holding a microchamber for hydrogel compression, which is achieved by moving a piston with 0.1 mm precision. In response to compression, concentration of the meshwork-conjugated kinase increases, whereas the excess liquid is extruded via the side walls constructed from a metal mesh. Using this system, spatial changes in collagen gel density can be observed via light scattering simultaneously with monitoring the status of kinase-phosphatase system within different areas of the hydrogel. The poster will describe construction and application of this experimental system to study the mechano-chemical response of this hybrid hydrogel. 2345-Pos Flow-Induced Self-Assembly of Spider Silk from Multi-Scale Simulations Ana M. Herrera1, Anil Kumar Dasanna2, Ulrich S. Schwarz3, Frauke Gr€ater4. 1 Molecular Biomechanics, Heidelberg Institute for Theoretical Studies gGmbH, Heidelberg, Germany, 2Heidelberg Univ, Heidelberg, Germany, 3 Inst Theoret Phys, Heidelberg Univ, Heidelberg, Germany, 4Heidelberg Institute for Theoretical Studies gGmbH, Heidelberg, Germany. Dragline spider silk proteins or spidroins self-assemble into an outstandingly tough fiber. The combination of ductility and strength relies in its microscopic structure within the fiber: small and strong beta-sheet crystals formed by polyalanine repeats embedded into a flexible amorphous matrix of glycine-rich repeats. Flow is a critical factor for the assembly of the highly disordered silk proteins into this elaborated structure within the fibril. However, the mechanism of flow-induced silk self-assembly remains elusive. We studied oligomer formation of tethered repetitive dragline peptides under uniform flow using non-equilibrium multi-scale molecular dynamics simulations at different flow rates. In atomistic Molecular Dynamics simulations, we found poly-alanine repeats to primarily drive the self-assembly, confirming crystal formation to promote fibrillation. We used this finding in more coarse-grained hydrodynamic simulations with aminoacid resolution, treating the silk proteins as semi-flexible block copolymers. We observed that medium to high flow velocities (>20 cm/s) increase alignment and crystallization of the silk peptides. High velocities (> 50 cm/s) result in extensions close to the contour length of the protein (>90%), thereby slowing down the assembly process as protein fluctuations are largely abolished. Our results yield a microscopic understanding of flowinduced silk assembly, which is likely relevant also for other flowdependent proteins. 2346-Pos Transformation of Tubulin Architectures by using Cationic Polymer as a Molecular Switch Juncheol Lee1, Chaeyeon Song1, Jimin Lee1, Herbert P. Miller2, Hasaeam Cho1, Bopil Gim1, Youli Li2, Stuart C. Feinstein2, Leslie Wilson2, Cyrus R. Safinya3, Myung Chul Choi1. 1 KAIST, Daejeon, Republic of Korea, 2Univ Calif, Santa Barbara, CA, USA, 3 Dept Matl Res, Univ Calif Santa Barbara, Santa Barbara, CA, USA. Tubulins heterodimers are pre-programmed biological building blocks. abtubulins are assembled into cytoskeleton microtubules (MTs), which are dynamic protein nanotubes involved in many important cellular functions. During the growth and shrinkage of MTs, the conformational changes of tubulin building blocks occur upon the hydrolysis of nucleotide bound to 479a tubulin. The tubulin assembly is known to be sensitive to various molecules such as MT-associated proteins (ex. Tau), drugs (ex. Taxol), and even cationic molecules (ex. divalent magnesium ions). We show our recent findings on the tubulin architectures in the presence of cationic polymers, in results which play the role as molecular switches. Structures of the assemblies were studied by using synchrotron small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). 2347-Pos Simulated Mechanical and Electrical Properties of Three-Dimensional Protein Lattices Rachel Baarda1, Simon Kit Sang Chu2, Tegan Marianchuk3, Daniel L. Cox1. 1 Physics, Univ Calif Davis, Davis, CA, USA, 2Biophysics, Univ Calif Davis, Davis, CA, USA, 3Biophysical Science, University of Chicago, Chicago, IL, USA. The chief goal in the field of nanomaterials is to create macroscopic structures whose properties are precisely engineered at the nanoscale. Proteinbased lattices are promising candidates for this goal since proteins naturally self-assemble and play a huge variety of functional roles in their native environment. We use molecular dynamics simulations to characterize the mechanical and electrical properties of several three-dimensional lattices designed from protein subunits. Simulations of infinite lattices are achieved by constructing a ‘‘unit cell’’ structure and applying periodic boundary conditions across all faces of the cell to tile it in 3-D space, allowing for explicit interactions between the structure and its periodic images. Each unit cell consists of a mechanically robust rod-like protein belonging to the BetaSolenoid protein family covalently bonded to a symmetric multi-mer; in this study, tetramers and dodecomers were used to construct tetrahedrally coordinated lattices, analogous to the zinc-blende crystal structure. Once these structures have been relaxed through energy minimization and constrained equilibration, the structure is strained by applying a prescribed deformation to the unit cell and the atoms it contains. We then analyze the resultant stress and polarization produced by this strain and extract the corresponding elastic and piezoelectric moduli, respectively. These material properties are useful measures of the viability of these protein lattice structures as functional biomaterials. 2348-Pos Highly Processive DNA Origami Nanoscale Motors Alisina Bazrafshan1, Travis Meyer2, Hanquan Su1, Joshua Brockman2, Selma Piranej1, Aaron Blanchard2, Khalid Salaita1,2, Yonggang Ke1,2. 1 Chemistry, Emory University, Atlanta, GA, USA, 2Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA, USA. Biological motor proteins that convert chemical energy into controlled nanomechanical motion are essential and shared among all branches of life. Accordingly, there is considerable interest in developing synthetic analogues. Among the synthetic motors created thus far, unipedal and bipedal DNA walkers that undertake discrete steps on RNA tracks have shown the greatest promise with applications spanning from biosensing to cargo transport and cargo sorting. Nonetheless, DNA-based walkersfall short because of their limited speed, low endurance, and the lack of preferred directionality which is ultimately due to the lack of coordination between individual DNA legs. Herein, we uncover the design principles for creating autonomous, unidirectional and processive DNA motors. By taking advantage of the DNA origami three-dimensional self-assembly technique, we created a library of DNA motors that allow testing of structure-function relationships at the nanoscale. We tested the role of DNA-leg density, polyvalency, geometric distribution, and chassis rigidity. Importantly, the work reveals that the local density of DNA legs and not the absolute polyvalency, is the most important parameter contributing to effective motion. Furthermore, an anisotropic rigid chassis is necessary for unidirectional motion. This led to a rod-shaped DNA nanomotor that linearly and autonomously translocates micron distances without intervention through a forcefield or patterned track. These findings provide the rules for creating cooperative and more sophisticated synthetic molecular motors, thus reducing the gap in capabilities between biological and synthetic motors.