A simple surface modification to generate atomically-flat and hydrophobic substrates for gliding assays with protein motors

In vitro gliding assay is a well-established assay for determining the activity of protein motors, such as actin-associated myosins and microtubule-associated kinesins and dyneins. In one of the conventional methods, protein motors are immobilized onto a nitrocellulose-coated coverslip and it propels actin filaments in the presence of ATP. Gliding assays also serve as the foundation for protein-motor-based nanotechnological devices such as biosensing and sorting. However, the preparation of nitrocellulose-coated coverslips is time-consuming and produces rough surfaces. Furthermore, the nitrocellulose film exhibits high background autofluorescence, which can be a problem in single-molecule measurements. Here, we investigated the use of hexamethyldisilazane (HMDS) to study actomyosin function and characterized its physical properties on glass coverslips and glass capillary tubes. We showed that the total preparation time to coat a coverslip with HMDS is <30 minutes, which is 1 order of magnitude faster than the >12-hour protocol for coating glass surfaces with nitrocellulose. In contrast to nitrocellulose film, HMDS vapor deposition is effortless and provides an atomically flat surface with low autofluorescence. In addition, HMDS does not interfere with myosin function, which is indicated by the similar actin gliding speed when compared with nitrocellulose. Our results show that HMDS vapor deposition is a more favorable surface treatment to nitrocellulose for in vitro gliding assay.

CHA-based DNA stochastic walker that traverses on cell membranes

DNA walker, imitating protein motors, is a class of nucleic acid nanodevices and can move along a precisely defined “track”. With promising future in materials and biotechnology, DNA walker has...

Information processing in biological molecular machines

ABSTRACTBiological molecular machines are enzymes that simultaneously catalyze two processes, one donating free energy and second accepting it. Recent studies show that most native protein enzymes have a rich stochastic dynamics of conformational transitions which often manifests in fluctuating rates of the catalyzed processes and the presence of short-term memory resulting from the preference of certain conformations. For arbitrarily complex stochastic dynamics of protein machines, we proved the generalized fluctuation theorem predicting the possibility of reducing free energy dissipation at the expense of creating some information stored in memory. That this may be the case has been shown by interpreting results of computer simulations for a complex model network of stochastic transitions. The subject of the analysis was the time course of the catalyzed processes expressed by sequences of jumps at random moments of time. Since similar signals can be registered in the observation of real systems, all theses of the paper are open to experimental verification.STATEMENT OF SIGNIFICANCEThe transient utilization of memory for storing information turns out to be crucial for the movement of protein motors and the reason for most protein machines to operate as dimers or higher organized assemblies. From a broader physical point of view, the division of free energy into the operation and organization energies is worth emphasizing. Information can be assigned a physical meaning of a change in the value of both these functions of state.

Tunable corrugated patterns in an active nematic sheet

Active matter locally converts chemical energy into mechanical work and, for this reason, it provides new mechanisms of pattern formation. In particular, active nematic fluids made of protein motors and filaments are far-from-equilibrium systems that may exhibit spontaneous motion, leading to actively driven spatiotemporally chaotic states in 2 and 3 dimensions and coherent flows in 3 dimensions (3D). Although these dynamic flows reveal a characteristic length scale resulting from the interplay between active forcing and passive restoring forces, the observation of static and large-scale spatial patterns in active nematic fluids has remained elusive. In this work, we demonstrate that a 3D solution of kinesin motors and microtubule filaments spontaneously forms a 2D free-standing nematic active sheet that actively buckles out of plane into a centimeter-sized periodic corrugated sheet that is stable for several days at low activity. Importantly, the nematic orientational field does not display topological defects in the corrugated state and the wavelength and stability of the corrugations are controlled by the motor concentration, in agreement with a hydrodynamic theory. At higher activities these patterns are transient and chaotic flows are observed at longer times. Our results underline the importance of both passive and active forces in shaping active matter and demonstrate that a spontaneously flowing active fluid can be sculpted into a static material through an active mechanism.