The interaction between the two heads of myosin II during motion and force production is poorly understood. To examine this issue, we developed an expression and purification strategy to isolate homogeneous populations of heterodimeric smooth muscle heavy meromyosins containing heads with different properties. As an extreme example, we characterized a heterodimer containing one native head and one head locked in a "weak binding" state by a point mutation in switch 2 (E470A). The in vitro actin

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... ilament motility of this heterodimer was the same as the homodimeric control with two cycling heads, suggesting that only one head of a pair actively interacts with actin to generate maximal velocity. A second naturally occurring heterodimer contained two cycling heads with 2-fold different activity, due to the presence or absence of a 7-amino acid insert near the active site. Enzymatically this (؉/؊) insert heterodimer was indistinguishable from a (50:50) mixture of the two homodimers, but its motility averaged 17% less than that of the mixture. These data suggest that one head of a heterodimer can disproportionately affect the mechanics of double-headed myosin, a finding relevant to our understanding of heterozygous mutant myosins found in disease states like familial hypertrophic cardiomyopathy. There has been speculation for many years about whether myosin derives any advantage from having two cross-bridge heads. The double-headed structure is clearly not necessary for actin binding or enzymatic activity, because it has long been known that proteolytically derived, single-headed subfragment 1 (S1) 1 retains both of these abilities (1). Moreover, a single-headed molecule can move actin filaments in the in vitro motility assay (2, 3). However, the velocities sustained by oneheaded fragments in this assay are generally less than that of double-headed species from the same myosin, and recent measurements in the laser trap have indicated that the unitary step size of a single-headed myosin is only half as great as that of a double-headed molecule (4). These observations indicate not only that two heads are more effective than one in moving actin but also that they may work together in some sort of coordinated fashion. One way to gain insight into the nature of potential headhead interactions is to study myosins containing two heads that differ functionally. If each head works independently, then the activity of such a heterodimer should be halfway between that of the faster homodimer and the slower homodimer. On the other hand, if there is an interaction between the heads, the properties of the heterodimer might be closer to that of one of the homodimeric species. To study heavy meromyosin (HMM) molecules with different heads, we developed an expression strategy that involves differential labeling of the constituent heavy chains with FLAG and His tags, co-infection in the Sf9 cell system, and sequential affinity chromatography columns to isolate homogeneous preparations. We assessed two types of smooth muscle HMM heterodimer using enzymatic and mechanical assays. The first was composed of two naturally occurring smooth muscle heavy chain isoforms that differ in vitro by a factor of two in their actinactivated ATPase activity and actin filament motility (5, 6). This difference is due to the presence or absence of a 7-amino acid insert in the surface loop near the nucleotide-binding pocket (7, 8), which has been called loop 1 (9). Such heterodimers almost certainly exist in nature, because the mRNAs for the ϩinsert and Ϫinsert heavy chains are co-expressed in single smooth muscle cells (10). The second heterodimer contained one wild type (WT) heavy chain and a second head with a mutation in switch 2 (E470A) that prevents the formation of a salt bridge between this residue and R247 in switch 1 of the active site. This mutation slows the ATPase activity of smooth myosin by two orders of magnitude, essentially "locking" it in a weak binding configuration (11, 12) . Thus, the heterodimeric E470A/WT molecule has a much greater disparity in function between the two heads. Enzymatic assays indicated that the two heads of both heterodimers function independently. However, the in vitro motility assay showed that the heterodimeric (ϩinsert/Ϫinsert) HMM moved actin filaments 17% more slowly than a 50:50 mixture of the corresponding homodimeric HMMs. In contrast, the heterodimeric E470A/WT HMM showed motility that was not significantly different from WT HMM. These results are consistent with the notion that myosin employs only one head at a time to perform work on the actin filament and, furthermore, suggest that one head of a heterodimer can exert a