During the 1980 s, the structures of actin and myosin were known only at the resolution shown in Figure $5-28$ a, b. Although researchers knew that the $\mathrm{S} 1$ portion of myosin bound to actin and hydrolyzed ATP, there was a substantial debate about where in the myosin molecule the contractile force was generated. At the time, two competing models were proposed for the mechanism of force generation in myosin. In the "hinge" model, S1 bound to actin, but the pulling force was generated by contraction of the "hinge region" in the myosin tail. The hinge region is in the heavy meromyosin portion of the myosin molecule, near where trypsin cleaves off light meromyosin (see Fig. $5-27$ b) ; this is roughly the point labeled "Two supercoiled $\alpha$ helices" in Figure $5-27$ a. In the "S1" model, the pulling force was generated in the S1 "head" itself and the tail was just for structural support. Many experiments were performed but provided no conclusive evidence. Then, in $1987,$ James Spudich and his colleagues at Stanford University published a study that, although not conclusive, went a long way toward resolving this controversy. Recombinant DNA techniques were not sufficiently developed to address this issue in vivo, so Spudich and colleagues used an interesting in vitro motility assay. The alga Nitella has extremely long cells, often several centimeters long and about $1 \mathrm{mm}$ in diameter. These cells have actin fibers that run along their long axes, and the cells can be cut open along their length to expose the actin fibers. Spudich and his group had observed that plastic beads coated with myosin would "walk" along these fibers in the presence of ATP, just as myosin would do in contracting muscle. For these experiments, the researchers used a more well-defined method for attaching the myosin to the beads. The "beads" were clumps of killed bacterial (Staphylococcus aureus) cells. These cells have a protein on their surface that binds to the Fc region of
antibody molecules (Fig. 5-21a). The antibodies, in turn, bind to several (unknown) places along the tail of the myosin molecule. When bead-antibody-myosin complexes were prepared with intact myosin molecules, they would move along Nitella actin fibers in the presence of ATP.
(a) Sketch a diagram showing what a bead-antibody-myosin complex might look like at the molecular level.
(b) Why was ATP required for the beads to move along the actin fibers?
(c) Spudich and coworkers used antibodies that bound to the myosin tail. Why would this experiment have failed if they had used an antibody that bound to the part of S1 that normally bound to actin? Why would this experiment have failed if they had used an antibody that bound to actin? To help focus on the part of myosin responsible for force production, Spudich and colleagues used trypsin to produce two partial myosin molecules (Fig. $5-27$ b): (1) heavy meromyosin (HMM), made by briefly digesting myosin with trypsin; HMM consists of S1 and the part of the tail that includes the hinge; and (2) short heavy meromyosin (SHMM), made from a more extensive digestion of HMM with trypsin; SHMM consists of S1 and a shorter part of the tail that does not include the hinge. Brief digestion of myosin with trypsin produces HMM and light meromyosin, by cleavage of a single specific peptide bond in the myosin molecule.
(d) Why might trypsin attack this peptide bond first rather than other peptide bonds in myosin?
Spudich and colleagues prepared bead-antibody-myosin complexes with varying amounts of myosin, HMM, and SHMM and measured their speed of movement along Nitella actin fibers in the presence of ATP. The graph below sketches their results.
(e) Which model ("S1" or "hinge") is consistent with these results? Explain your reasoning.
(f) Provide a plausible explanation for the increased speed of the beads with increasing myosin density.
(g) Provide a plausible explanation for the plateauing of the speed of the beads at high myosin density.
The more extensive trypsin digestion required to produce SHMM had a side effect:
another specific cleavage of the myosin polypeptide backbone in addition to the cleavage in the tail. This second cleavage was in the S1 head.
(h) Why is it surprising that SHMM was still capable of moving beads along the actin fibers?
(i) As it turns out, the tertiary structure of the $\mathrm{S} 1$ head remains intact in SHMM. Provide a plausible explanation of how the protein remains intact and functional even though the polypeptide backbone has been cleaved and is no longer continuous.