Mechanical properties of single myosin molecules probed with the photonic force microscope

Abstract

To characterize elastic properties and geometrical parameters of individual, whole myosin molecules during their interaction with actin we sparsely adsorbed myosin molecules to nanometer-sized microspheres. Thermally driven position fluctuations of these microspheres were recorded with the three-dimensional detection scheme of the photonic force microscope. Upon binding of single myosin molecules to immobilized actin filaments in the absence of ATP, these thermally driven position fluctuations of the microspheres change significantly. From three-dimensional position fluctuations stiffness and geometrical information of the tethering molecule can be derived. Axial stiffness was found to be asymmetric, approximately 0.04 pN/nm for extension, approximately 0.004 pN/nm for compression. Observed stiffness of whole myosin molecules is much less than estimated for individual myosin heads in muscle fibers or for single-molecule studies on myosin fragments. The stiffness reported here, however, is identical to stiffness found in other single-molecule studies with full-length myosin suggesting that the source of this low stiffness is located outside the myosin head domain. Analysis of geometrical properties of tethering myosin molecules by Brownian dynamics computer simulations suggests a linker length of approximately 130 nm that is divided by a free hinge located approximately 90 nm above the substrate. This pivot location coincides with myosin's hinge region. We demonstrate the general applicability of thermal fluctuation analysis to determine elastic properties and geometrical factors of individual molecules.

FIGURE 1

Relations between thermally driven position fluctuations of a tethered bead, probability distribution of bead position, free energy profiles, and force functions along x (linear elasticity assumed) and along z (linear but asymmetric elasticity). (A) Schematic drawing of a microsphere tethered to an actin filament by a single myosin molecule. (B) Time course of bead position along x. (C) Plots of probability distribution of bead position along x, free energy profile, and force function with respect to x. (D) Force function F(z), free energy profile E(z), and probability distribution P(z) for position fluctuations along z.

FIGURE 2

Raw data of an approach of a trapped bead with myosin II molecules in very low density on its surface. For further details see text.

FIGURE 3

Surfaces of equal position probability (95% probability) derived from position fluctuations recorded during approaches such as that shown in Fig. 2. (A) Ellipsoid-like shape of a freely fluctuating bead in a very weak laser trap, e.g., during period A in Fig. 2. (B) New shape of surface of equal position probability after sudden reduction in thermal fluctuation along the z axis, e.g., in period B of Fig. 2. (C) “Cut-off” ellipsoid observed when myosin-free microspheres are moved against a BSA-coated surface with or without immobilized actin filaments.

FIGURE 4

(A) Two-dimensional (x, z), color-encoded energy landscape derived from the three-dimensional probability distribution of the tethered microsphere shown in Fig. 3 B. (B) Asymmetric one-dimensional energy profile along the z axis (black solid line) in the energy landscape shown in A. The spring constants away (as) and toward (ts) the actin filament were fitted to the experimental data (+) using two different harmonic fits starting at the minimum of the energy profile.

FIGURE 5

Distribution of axial spring constants away from the surface, i.e., away from the actin filament. Data of 63 myosin molecules tethering a microsphere to an actin filament immobilized on a glass surface.

FIGURE 6

Extension of the range of bead fluctuations (A) along and (B) across the actin filament after the tether had formed. Isoprobability surfaces (95% probability) of the three-dimensional probability distributions of the tethered bead are shown for four different positions along the actin filament axis (x axis; A) and for five different positions across the actin filament axis (y axis; B). The isoprobability surface located at the origin of the coordinates is the surface observed at the position of the bead when the tether had formed. The ellipsoid illustrated by the solid line represents the isoprobability surface observed for the free bead fluctuating in the optical trap just before the tether had formed. (C) Image of a fluorescently labeled microsphere of 500 nm in diameter bound in an ATP-free solution to a single TRITC-phalloidin labeled, biotinylated actin filament immobilized on a neutravidin-coated glass surface.

FIGURE 7

Two-dimensional color-encoded energy landscapes derived from slices through the probability distributions shown in Fig. 6 B. Slices were performed along the planes as indicated in Fig. 6 B, except for A, which is the energy landscape resulting form a cut parallel to the glass surface at a z-position of ∼20 nm.

FIGURE 8

Schematic drawing of the location of the pivot and the effective linker length derived from Brownian dynamics simulations. Experimental data (shown in Fig. 7 C) superimposed as line profiles onto data derived from Brownian dynamics simulations (shown in gray scale). The absolute altitude of the bead position above the surface was derived from the lowest position at the leftmost end of the distribution. For a 500-nm bead this position is 250 nm above the substrate surface.

FIGURE 9

(A) Brightness-encoded two-dimensional energy landscape of a BSA-coated bead (no myosin) derived from three-dimensional probability distributions. When the bead was moved toward a neutravidin-coated, BSA-blocked surface the accessible space became restricted (see Fig. 3 C). (B) Energy profile along the black line in A that is perpendicular to the substrate surface. Experimental data are represented by crosses, whereas data from Brownian dynamics simulations appear as a solid line.

FIGURE 10

Schematic drawing of a myosin molecule tethering a microsphere to an immobilized actin filament. Names of proteolytic fragments (S-1, S-2, HMM, and LMM) as well as distances between bending points within a myosin molecule (Elliott and Offer, 1978; Walker et al., 1985) are indicated in black.