Position-Marking of Myosin Heads with Site-Directed Antibodies
Since the hydrated specimens are observed in unstained conditions, it is absolutely necessary to position-mark individual myosin head in myosin filaments by an appropriate means. For this purpose, we used three different site-directed antibodies (antibodies 1-3) to myosin head (Sutoh et al., 1989; Minoda et al., 2011). Antibody 1 (anti-CAD antibody) attaches to junctional peptide between 50 and 20 k segments of myosin heavy chain in myosin head catalytic domain (CAD), antibody 2 (anti-RLR antibody) attaches to peptides around reactive lysine residue (Lys83) in myosin head converter domain (COD), and antibody 3 (antiLD antibody) attaches to two regulatory light chains in myosin head lever arm domain (LD). Figure 1.8 is a ribbon diagram showing structure of myosin head, in which the sites of attachment of antibodies 1, 2, and 3 are indicated by numbers 1, 2 and 3 and 3', respectively, while Fig. 1.9 shows electron micrographs of rotary shadowed myosin molecules with antibodies (IgG) attached (Sutoh et al., 1989; Minoda et al., 2011).
Figure 1.8 Ribbon diagram of myosin head showing approximate attachment regions of antibodies 1, 2, and 3, indicated by numbers 1, 2, and 3 and 3', respectively. Catalytic domain consists of 25 K (green), 50 K (red), and part of 20 K (dark blue) fragments of myosin heavy chain, while lever arm domain (LD) consists of the rest of 20 K fragment and essential (ELC, light blue) and regulatory (RLC, magenta) light chains. CAD and LD are connected via small converter domain (COD). Location of peptides around Lys83, and that of two peptides (Met58~Ala70, and Leu106 ~Phe120 in LD are colored yellow. From Minoda et al. (2011).
Individual myosin heads were position-marked with colloidal gold particles (diameter, 20 nm; coated with protein A, working as a paste to connect proteins, (EY laboratories) via one of the three antibodies. Since native myosin filaments are too thin and tend to curl and aggregate, we used synthetic myosin filaments (myosin- myosin rod cofilaments), prepared by polymerizing myosin and myosin rod molecules at low ionic strength (Sugi et al., 1997). As shown in Fig. 1.10, spindle-shaped synthetic myosin filaments,
with a number of gold particles attached to individual myosin
heads, can be recorded with the imaging plate (IP).
Figure 1.9 A gallery of electron micrographs of antibody 1,2 or 3(IgG)- myosin head complexes. IgG molecules are indicated by arrowheads. From Minoda et al. (2011).
Figure 1.10 (a, b) Typical imaging plate (IP) records of bipolar myosin-
myosin rod cofilaments with a number of gold particles attached to individual myosin heads. (c) Enlarged view of the filaments shown in b. From Sugi et al. (2008).
Recording of Specimen Image and Data Analysis
Under a magnification of 10,000x, the average number of electrons during the exposure time (0.1 s) was ~10. Reflecting this electron statistics, the image of each gold particle on the IP consisted of 20-50 dark pixels with different gradation. The center of mass position for each particle was determined as the coordinates (two significant figures; accuracy, 0.6 nm) within a single pixel where the center of mass position was located. These coordinates representing the particle position was taken as the position of myosin head. The change in position of myosin head was compared between the two IP records of the same myosin filament (Sugi et al., 1997, 2008).