The widespread use of controlled molecular-level motion in key natural processes suggests that great rewards could come from bridging the gap between the present generation of synthetic molecular systems, which by and large rely upon electronic and chemical effects to carry out their functions, and the machines of the macroscopic world, which utilize the synchronized movements of smaller parts to perform specific tasks. This is a scientific area of great contemporary interest and extraordinary recent growth, yet the notion of molecular-level machines dates back to a time when the ideas surrounding the statistical nature of matter and the laws of thermodynamics were first being formulated. Here we outline the exciting successes in taming molecular-level movement thus far, the underlying principles that all experimental designs must follow, and the early progress made towards utilizing synthetic molecular structures to perform tasks using mechanical motion. We also highlight some of the issues and challenges that still need to be overcome.

Synthetic molecular motors and mechanical machines.

The present invention relates to a structure comprising a biological membrane and a porous or perforated substrate, a biological membrane, a substrate, a high throughput screen, methods for production of the structure membrane and substrate, and a method for screening a large number of test compounds in a short period. More particularly it relates to a structure comprising a biological membrane adhered to a porous or perforated substrate, a biological membrane capable of adhering with high resistance seals to a substrate such as perforated glass and the ability to form sheets having predominantly an ion channel or transporter of interest, a high throughput screen for determining the effect of test compounds on ion channel or transporter activity, methods for manufacture of the structure, membrane and substrate, and a method for monitoring ion channel or transporter activity in a membrane.

High throughput screen

Cells employ a variety of linear motors, such as myosin, kinesin and RNA polymerase, which move along and exert force on a filamentous structure. But only one rotary motor has been investigated in detail, the bacterial flagellum (a complex of about 100 protein molecules). We now show that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion. A central rotor of radius approximately 1 nm, formed by its gamma-subunit, turns in a stator barrel of radius approximately 5nm formed by three alpha- and three beta-subunits. F1-ATPase, together with the membrane-embedded proton-conducting unit F0, forms the H+-ATP synthase that reversibly couples transmembrane proton flow to ATP synthesis/hydrolysis in respiring and photosynthetic cells. It has been suggested that the gamma-subunit of F1-ATPase rotates within the alphabeta-hexamer, a conjecture supported by structural, biochemical and spectroscopic studies. We attached a fluorescent actin filament to the gamma-subunit as a marker, which enabled us to observe this motion directly. In the presence of ATP, the filament rotated for more than 100 revolutions in an anticlockwise direction when viewed from the 'membrane' side. The rotary torque produced reached more than 40 pN nm(-1) under high load.

Direct observation of the rotation of F1-ATPase

Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble averaging. Distributions and time trajectories of these observables can therefore be measured during a reaction without the impossible need to synchronize all the molecules in the ensemble. The progress in applying these tools to biological studies with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.

https://science.sciencemag.org/content/sci/283/5408/1676.full.pdf

Fluorescence spectroscopy of single biomolecules.

By introducing twist during spinning of multiwalled carbon nanotubes from nanotube forests to make multi-ply, torque-stabilized yarns, we achieve yarn strengths greater than 460 megapascals. These yarns deform hysteretically over large strain ranges, reversibly providing up to 48% energy damping, and are nearly as tough as fibers used for bulletproof vests. Unlike ordinary fibers and yarns, these nanotube yarns are not degraded in strength by overhand knotting. They also retain their strength and flexibility after heating in air at 450°C for an hour or when immersed in liquid nitrogen. High creep resistance and high electrical conductivity are observed and are retained after polymer infiltration, which substantially increases yarn strength.

http://science.sciencemag.org/content/sci/306/5700/1358.full.pdf

Multifunctional Carbon Nanotube Yarns by Downsizing an Ancient Technology

Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope

Filamentous structures are abundant in cells. Relatively rigid filaments, such as microtubules and actin, serve as intracellular scaffolds that support movement and force, and their mechanical properties are crucial to their function in the cell. Some aspects of the behaviour of DNA, meanwhile, depend critically on its flexibility—for example, DNA-binding proteins can induce sharp bends in the helix1. The mechanical characterization of such filaments has generally been conducted without controlling the filament shape, by the observation of thermal motions2,3,4,5 or of the response to external forces6,7,8,9 or flows10,11,12. Controlled buckling of a microtubule has been reported13, but the analysis of the buckled shape was complicated. Here we report the continuous control of the radius of curvature of a molecular strand by tying a knot in it, using optical tweezers to manipulate the strand's ends. We find that actin filaments break at the knot when the knot diameter falls below 0.4 µm. The pulling force at breakage is around 1 pN, two orders of magnitude smaller than the tensile stress of a straight filament. The flexural rigidity of the filament remained unchanged down to this diameter. We have also knotted a single DNA molecule, opening up the possibility of studying curvature-dependent interactions with associated proteins. We find that the knotted DNA is stronger than actin.

/pdf/tying-a-molecular-knot-with-optical-tweezers-1eho22ibtp.pdf

Tying a molecular knot with optical tweezers

The unbinding and rebinding of motor proteins and their substrate filaments are the main components of sliding movement We have measured the unbinding force between an actin filament and a single motor molecule of muscle, myosin, in the absence of ATP, by pulling the filament with optical tweezers The unbinding force could be measured repeatedly on the same molecule, and was independent of the number of measurements and the direction of the imposed loads within a range of +/- 90 degrees The average unbinding force was 92 +/- 44 pN, only a few times larger than the sliding force but an order of magnitude smaller than other intermolecular forces From its kinetics we suggest that unbinding occurs sequentially at the molecular interface, which is an inherent property of motor molecules

Unbinding force of a single motor molecule of muscle measured using optical tweezers

http://www.k2.phys.waseda.ac.jp/PDF/1996JMB_Yasuda_FAtors.pdf

Hidetake Miyata

Papers

Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope

Tying a molecular knot with optical tweezers

Unbinding force of a single motor molecule of muscle measured using optical tweezers

Direct Measurement of the Torsional Rigidity of Single Actin Filaments

Real time imaging of single fluorophores on moving actin with an epifluorescence microscope