In recent years the structural basis of the actomyosin crossbridge cycle 1 has been defined in increasing detail by X-ray crystallography and electron microscopy 2-9 . 2 However, while crystal structures of myosin motor domains have been obtained for a number of nucleotide-bound states, the nature of the actin binding interaction has not been observed directly to date 2,3,8,10,11 . Residues involved in actin binding were identified by fitting the envelope of the myosin crystal structure into the electron micrograph density of decorated actin filaments. Initial docking attempts suggested that the cleft between the upper and lower 50K myosin domains might close on forming the rigor state 12 . This approach has been refined to the point of identifying a potential hinge point around switch 1 at the myosin nucleotide site that allows the upper 50K domain to rotate towards the lower 50K domain 7 . Recent crystallographic studies support this concept 6,8,9 . While structural evidence is mounting in favour of the cleft closure model, it is important to test this idea in solution. Structural data alone provides only a static picture and the proposed transitions between states have been made by inference. We have introduced cysteine residues at position 416 and 537 across the cleft (Dictyostelium discoideum, Dd, myosin II sequence) in a cysteine-deficient 13 myosin background (Fig 1a,b). Labelling these cysteine residues with N-1-pyrene

An extensive series of experiments in this laboratory has shown that the binding of actin to rabbit skeletal muscle myosin subfragment-1 (a single-headed subfragment) can be described by a two-step model, with formation of a weakly bound complex, the A-state, followed by an isomerization to a more tightly bound complex, the R-state. In this paper, we report on additional experiments comparing the subfragment-1 with heavy meromyosin (a two-headed subfragment). Using a modeling approach, we have quantitated the two-step binding for each of the two heads. This indicates that the binding is cooperative and leads to a more complex view of the acto-myosin interaction than has previously been acknowledged. Implications for the dynamic behavior of the two heads during muscle contraction are discussed.

Total internal reflection fluorescence (TIRF) microscopy, used in conjunction with flash photolysis, provides a useful way of investigating the kinetics of macromolecular interactions. We compare different TIRF optical geometries to establish an optimal combination. Excitation light was introduced via four different arrangements: (1) a prism positioned on the microscope optical axis, (2) an offset prism with propagation through a silica slide trans to the objective lens, (3) an offset prism with propagation through a silica coverslip cis to a water‐immersion objective lens and (4) a prismless arrangement using a high NA oil‐immersion objective lens. Photolysis was achieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sample. Although the offset trans prism gave the best signal‐to‐background ratio, a customised thin rhombic prism incorporated, on axis, into the flash condenser assembly was almost as good and was more practical for scanning multiple fields. An oil‐immersion lens gave the brightest image for sample depths < 30 µm but above this limit, a water‐immersion lens was better. The prismless arrangement may offer advantages in other situations but it is important to check the actual numerical aperture of the objective lens.

The fluorescence properties of Dictyostelium discoideum (Dd) myosin II constructs containing a single tryptophan residue have revealed detailed information regarding nucleotide binding and hydrolysis steps. Here we extend these studies to investigate the influence of actin on nucleotide-induced fluorescence transients. The fluorescence from native actin tryptophan residues is not significantly perturbed on binding to myosin, although an apparent signal is detected as a consequence of a light scatter artifact. Actin has a minor effect on the response of W129, located at the entrance to the nucleotide-binding pocket, and reduces the forward rate constants for the isomerization(s) associated with binding of ATP, ATPgammaS, and ADP by 3-fold or less. The isomerization detected by W129 clearly precedes the dissociation of actin in the case of ADP and ATPgammaS binding. The fluorescence from the conserved W501 residue, located at the distal end of the relay helix, is very sensitive to the switch 2 and/or lever arm disposition. Consequently, the observed fluorescence emission intensity can be used to estimate the equilibrium constant between the pre- and post-power stroke conformations. Actin modulates this equilibrium by no more than 2-fold in the presence of nucleoside triphosphate. These data have implications for the mechanism of product release and suggest that actin activates another process in the mechanism, such as switch 1 movement and Pi release, rather than influencing the switch 2 equilibrium and lever arm position directly.

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