Detection of single-molecule interactions using correlated thermal diffusion

Abstract

Observation of discrete, single-molecule binding events allows one to bypass assumptions required to infer single-molecule properties from studies of ensembles of molecules. Optically trapped beads and glass microneedles have been applied to detect single-molecule binding events, but it remains difficult to identify signs of binding events given the large displacements induced by thermal forces. Here, we exploit thermal diffusion by using correlation between motion of optically trapped beads attached to both ends of a single actin filament to track binding events of individual myosin molecules. We use correlated diffusion to measure the stiffness of a single myosin molecule and estimate its thermal fluctuation in a poststroke state as comparable in amplitude to the measured stroke distance. The use of correlated diffusion to measure kinetics of single-molecule interactions and the stiffness of the interacting moieties should be applicable to any pair of interacting molecules, and not limited to biological motors.

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

“…If the stiffness of the acto-myosin structure is dominated by cantilever bending of the lever arm, then stiffness should decrease with lever arm length. The stiffness we measure for acto-M10HMM is consistent with this idea because it lies between previous values measured for the shorter stiffer two IQ myosin-2 lever arm (∼0.7-1.7 pN/nm) (31,32,41,63,64) and the longer and more compliant six IQ myosin-5a lever arm (∼0.2 pN/nm) (38). In addition, single-headed M10HMM binding to F-actin observed via EM (Fig.…”

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

“…However, we cannot cover articles in languages we cannot read; articles completely in languages other than English, French, German, Italian, and Spanish are therefore not included (not included but on-topic articles in Japanese, Chinese, or Korean: Hozawa, 1911;Sheng et al, 1956;Yazawa and Yoshida, 1979;Fan and Chen, 1986;Nishita, 1998;Katoh, 1999;Ojima, 2003;Funabara, 2004;in Russian: Samosudova and Frank, 1962;Razumova et al, 1966Razumova et al, , 1968Razumova et al, , 1972Razumova et al, , 1973aRazumova et al, , 1973bRazumova et al, , 1975. We have also not included articles in which the species used is not identified or cannot now be identified (requiring a book long out of print) (Aubert, 1944;Kuschinski and Turba, 1950;Szent-Györgyi, 1953;Philpott and Szent-Györgyi, 1954;Szent-Györgyi and Borbiro, 1956;Cohen and Szent-Györgyi, 1957;Szent-Györgyi and Cohen, 1957;Szent-Györgyi et al, 1960;Maruyama and Ishikawa, 1963;Shechter and Blout, 1964;Baier and Zobel, 1966;Kominz and Maruyama, 1967;Botts et al, 1972;Moos, 1972;Ogawa, 1985;Mehta et al, 1997).…”

The power stroke of myosin VI and the basis of reverse directionality

“…Motor dilutions were chosen to give conditions in which Ϸ10% of tested platforms yielded actomyosin binding events. Binding events were identified in the data traces by eye using the drop in position variance and bead-bead correlation (21).…”

“…An actin filament is held between two optically trapped beads and suspended over a platform until isolated interactions are observed between the filament and a single myosin molecule. Attachment of actin to surface-bound myosin can be detected by correlation of the Brownian motion of the trapped beads (21). The attachment time ⌬t and positional displacement of the beads from equilibrium ⌬x are then measured for each interaction event.…”

“…Displacements are shown before and after correction for series compliance. Correction factors were estimated using the method of correlated thermal diffusion (21). For comparison, gliding velocities V for each construct are also tabulated (mean Ϯ SD; see Fig.…”