Biomedical subjects

Fa-Qing Zhao

Publications and source records attributed to Fa-Qing Zhao.

3 recordsLinked to original sources

PubMed2005

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. These studies suggest that the OFF state is achieved by an asymmetric, intramolecular interaction between the actin-binding region of one head and the converter region of the other, switching both heads off. Although this is a plausible model for relaxation based on isolated myosin molecules, it does not reveal whether this structure is present in native myosin filaments. Here we analyse the structure of a phosphorylation-regulated striated muscle thick filament using cryo-electron microscopy. Three-dimensional reconstruction and atomic fitting studies suggest that the 'interacting-head' structure is also present in the filament, and that it may underlie the relaxed state of thick filaments in both smooth and myosin-regulated striated muscles over a wide range of species.

Animals↗

PubMed2003

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+. The structural change is thus a result of Ca2+ binding alone and not an artifact of staining. Comparison of the two techniques suggests that negative staining preserves the structure induced by Ca2+ -binding. We therefore used a time-resolved negative staining technique to determine the time scale of the structural change. Full disordering was observed within 30 ms of Ca2+ addition, and had started to occur within 10 ms, showing that the change occurs on the physiological time scale. Comparison with studies of single heavy meromyosin molecules suggests that an increased mobility of myosin heads induced by Ca2+ binding underlies the changes in filament structure that we observe. We conclude that the loosening of the array of myosin heads that occurs on activation is real and physiological; it may function to make activated myosin heads freer to contact actin filaments during muscle contraction.

Actin Cytoskeleton↗

PubMed2003

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. The procedure consists of briefly (milliseconds) exposing the macromolecular structure of interest on an EM grid to conditions that initiate the structural change, then immediately fixing with uranyl acetate or tannic acid. Specimens are then observed by negative staining. The key finding that validates this approach is our demonstration that uranyl acetate, and in some cases tannic acid, fixes protein molecular structure on the millisecond timescale. This is demonstrated by our observation that exposure of actin and myosin filaments to these fixatives for as little as 10 ms is sufficient to fully preserve them against changes that normally induce rapid and major alteration in their molecular structure. Fixation appears to stabilize both ionic and hydrophobic bonds. This approach should be of general utility for studying transient molecular changes in many systems.

Actins↗