An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits.

Single particle analysis is widely used for three-dimensional reconstruction of helical filaments. Near-atomic resolution has been obtained for several well-ordered filaments. However, it is still a challenge to achieve high resolution for filaments with flexible subunits and a large axial rise per subunit relative to pixel size.

Structural basis of the relaxed state of a Ca2+-regulated myosin filament and its evolutionary implications.

Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca(2+) binding to the essential light chains, contributing to on-off switching or modulation of contraction. Phosphorylation-regulated filaments in the relaxed state are characterized by an asymmetric interaction between the two myosin heads, inhibiting their actin binding or ATPase activity. Here, we have tested whether a similar interaction switches off activity in myosin filaments regulated by Ca(2+) binding.

Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms.

Background: Tarantula has been used as a model system for studying skeletal muscle structure and function, yet data on the genes expressed in tarantula muscle are lacking.

Results: We constructed a cDNA library from Aphonopelma sp. (Tarantula) skeletal muscle and got 2507 high-quality 5'ESTs (expressed sequence tags) from randomly picked clones.

Head-head interaction characterizes the relaxed state of Limulus muscle myosin filaments.

Regulation of muscle contraction via the myosin filaments occurs in vertebrate smooth and many invertebrate striated muscles. Studies of unphosphorylated vertebrate smooth muscle myosin suggest that activity is switched off through an intramolecular interaction between the actin-binding region of one head and the converter and essential light chains of the other, inhibiting ATPase activity and actin interaction. The same interaction (and additional interaction with the tail) is seen in three-dimensional reconstructions of relaxed, native myosin filaments from tarantula striated muscle, suggesting that such interactions are likely to underlie the off-state of myosin across a wide spectrum of the animal kingdom.

Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity.

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs).

Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state.

Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electron microscopy to study the effects of blebbistatin on the organization of the myosin heads on muscle thick filaments. Loss of ADP and Pi from the heads causes thick filaments to lose their helical ordering.

Millisecond time-resolved changes occurring in Ca2+-regulated myosin filaments upon relaxation.

Contraction of many muscles is activated in part by the binding of Ca(2+) to, or phosphorylation of, the myosin heads on the surface of the thick filaments. In relaxed muscle, the myosin heads are helically ordered and undergo minimal interaction with actin. On Ca(2+) binding or phosphorylation, the head array becomes disordered, reflecting breakage of the head-head and other interactions that underlie the ordered structure.

Atomic model of a myosin filament in the relaxed state.

Contraction of muscle involves the cyclic interaction of myosin heads on the thick filaments with actin subunits in the thin filaments. Muscles relax when this interaction is blocked by molecular switches on either or both filaments. Insight into the relaxed (switched OFF) structure of myosin has come from electron microscopic studies of smooth muscle myosin molecules, which are regulated by phosphorylation.

Ca2+ causes release of myosin heads from the thick filament surface on the milliseconds time scale.

We have used electron microscopy to study the structural changes induced when myosin filaments are activated by Ca2+. Negative staining reveals that when Ca2+ binds to the heads of relaxed Ca2+ -regulated myosin filaments, the helically ordered myosin heads become disordered and project further from the filament surface. Cryo-electron microscopy of unstained, frozen-hydrated specimens supports this finding, and shows that disordering is reversible on removal of Ca2+.

Capturing time-resolved changes in molecular structure by negative staining.

Imaging structural intermediates of biological processes is a key step in understanding biological function. Because intermediates are commonly short-lived, lasting only milliseconds, the main methods used to capture them have been conventional imaging of analog or inhibited states, having extended lifetimes, or rapid (millisecond timescale) freezing of intermediates with subsequent observation by cryo-EM. We have developed a simpler method that fixes structure on the millisecond timescale.