To better understand the molecular basis for some of the unique mechanical properties of tonic smooth muscle, we use a laser trap to assay the mechanochemistry of single smooth muscle heavy meromyosin molecules lacking a seven-amino acid insert in the nucleotide binding loop (minus insert). We measured a second-order ATP-induced actin dissociation rate, k T , of 2. , and an ADP affinity, K D , of 3.2 M, which is more than 100-fold greater than that measured for skeletal muscle myosin. By performing in vitro motility studies under nearly identical conditions, we show that the relatively slow actin velocity generated by minus-insert heavy meromyosin is significantly influenced, but not limited, by k ؊D . Our results support a model in which two separate intermediate steps in the actin-myosin catalyzed ATP hydrolysis reaction are energetically coupled through mechanical interactions, and we discuss this model in the context of the ability of tonic muscle to maintain high forces at low energetic cost (latch).Muscle shortening and force generation result from actinmyosin binding events that are coupled to the actin-myosin catalyzed ATP hydrolysis reaction illustrated in Fig. 1. Upon binding to an actin filament (A) and releasing inorganic phosphate (P i ), myosin undergoes a large and discrete rotation of its lever-like light chain domain, which is capable of generating both motion and force (1-5). With the subsequent release of ADP (at the rate k ϪD ) an additional rotation of the light chain domain of myosin has been observed in smooth muscle myosin (6, 7), but unlike the work generating rotation associated with actin binding/P i release, the rotation associated with ADP release is thought to be a strain-sensing biochemical step (8 -10). Following the release of ADP, ATP binding induces the detachment of myosin from the actin filament (at the rate k T ), after which ATP is hydrolyzed.Muscles differ significantly in their shortening speeds and force-generating capacities. For instance, smooth muscle produces a greater average force per myosin and slower speeds of shortening than skeletal muscle (11). To a large extent these mechanical differences are caused by kinetic differences among the different myosin isoforms that exist within different muscle types (12, 13). For example, phasic and tonic smooth muscle (found in the intestine and aorta respectively) express two myosin heavy chain isoforms that differ by a seven-amino acid insert in a surface loop spanning their nucleotide binding pocket (14 -16). Phasic smooth muscle contains primarily the plus-insert myosin, whereas tonic smooth muscle contains primarily the minus-insert myosin. In addition, two essential light chain isoforms are coordinately expressed with the heavy chain isoforms. The acidic isoform (LC 17a ) is coexpressed with the plus-insert heavy chain whereas the basic isoform (LC 17b ) coexpresses with the minus-insert heavy chain (17-19). Based on in vitro motility studies, the presence or absence of the sevenamino acid insert in the heavy chain is t...

Myosin II has two heads that are joined together by an alpha-helical coiled-coil rod, which can separate in the region adjacent to the head-rod junction (Trybus, K. M. 1994. J. Biol. Chem. 269:20819-20822). To test whether this flexibility at the head-rod junction is important for the mechanical performance of myosin, we used the optical trap to measure the unitary displacements of heavy meromyosin constructs in which a stable coiled-coil sequence derived from the leucine zipper was introduced into the myosin rod. The zipper was positioned either immediately after the heads (0-hep zip) or following 15 heptads of native sequence (15-hep zip). The unitary displacement (d) decreased from d = 9.7 +/- 0.6 nm for wild-type heavy meromyosin (WT HMM) to d = 0.1 +/- 0.3 nm for the 0-hep zip construct (mean +/- SE). Native values were restored in the 15-hep zip construct (d = 7.5 +/- 0.7 nm). We conclude that flexibility at the myosin head-rod junction, which is provided by an unstable coiled-coil region, is essential for optimal mechanical performance.

Cryo-EM 3D reconstruction has paved the way for atomic resolution structure determination of noncrystalline biological samples (1). For practical purposes, the presence of symmetry and homogeneity among others are extremely helpful. One type of specimen where cryoEM has made major contributions is the structure of filaments with helical symmetry, including the recent atomic resolution structures of Factin alone (2) and when decorated with individual myosin heads (3). However, actin decorated with the double-headed myosin fragment, known as HMM, only shows helical symmetry out to approximately the end of the myosin motor domain (10 nm radius). The rest of the myosin head consists of a small, folded domain known as the "converter" and a long α-helix that binds a pair of light chains, dubbed essential (ELC), and regulatory (RLC). The lever arm amplifies small conformational changes in the myosin motor domain into a large axial movement at the end of the myosin head (4). The lever arms (extending a further 30 Å) cannot follow the F-actin symmetry unless the myosin coiled coil, here called the proximal S2, unfolds to accommodate the ~75 Å separation of the head-tail junctions. If the proximal S2 does not unfold, then the two lever arms must assume different structures which will impart some strain into the system and break the helical symmetry thus removing the most powerful feature of helical filaments for structure determination. Structures of myosin heads bound to actin in situ in muscle show the two lever arms coming together to a common origin rather than indicating separation due to unfolding of the proximal S2 (5, 6) at low resolution. The structure of F-actin decorated with HMM has only been investigated once previously using negative stain and imposition of helical symmetry (7). A structure of two-headed myosin II binding to F-actin is important for a mechanical description of force production in muscle. Here we use single particle reconstruction to test a protocol for avoiding imposition of helical symmetry and use frozen-hydrated specimens for improving specimen preservation and thereby assess prospects for high resolution structure determination of HMM decorated actin.