FIGURE 8.

cMyBP-C^C10mut has lost C10 domain interaction with myosin LMM. A, SDS-PAGE analysis followed by Coomassie Blue staining demonstrated the purity of His-tagged recombinant C8-C10^WT, C8-C10^mut, and myosin LMM proteins. B, various concentrations of C8-C10 recombinant proteins were used to show purity using anti-C10^WT rabbit antibodies and anti-C10^mut monoclonal antibodies. Ponceau staining was used to verify equal loading on the nitrocellulose membrane. C, prior to the co-sedimentation assay, recombinant proteins were individually tested for their sedimentation capacity. Net protein content before centrifugation (total (T)) and after centrifugation in the supernatants (S) and pellets (P) was determined by SDS-PAGE and Coomassie Blue staining of the gels. D, co-sedimentation of either C8-C10^WT or C8-C10^mut with LMM proteins. All protein concentrations were 6 μm in the assay at 250,000 × g for 10 min at 4 °C. Co-sedimentation between C8-C10^WT and LMM indicates interaction, as illustrated by the presence of C8-C10^WT in the pellet, whereas C8-C10^mut showed little binding. E, quantitative data summarized the amount of C8-C10^WT and C8-C10^mut proteins in the supernatants (S) and pellets (P) after centrifugation (n = 3). *, p < 0.001. F, a representative standard curve showing a range of known molar concentrations (4–40 pmol) of C8-C10^WT to myosin LMM (40 pmol) on the x axis that were blotted against the SDS-PAGE band intensity on the y axis. G, co-sedimentation assay was performed with varying concentration (1–30 μm) of either C8-C10^WT or C8-C10^mut to 5 μm of myosin LMM to determine the K_d and B_max values. The K_d and B_max values for C8-C10^WT (circles) were of 9.8 ± 1.8 and 1.14 ± 0.08 mol/mol (n = 5, means±S.E.). Because C8-C10^mut (squares) did not bind to myosin LMM, no significant K_d and B_max values could be determined (n = 5, means ± S.E.). H, cross-linking analysis of either C8-C10^WT or C8-C10^mut with LMM showed cross-linking of C8-C10^WT with LMM (arrow), indicating interaction, whereas C8-C10^mut did not cross-link to LMM. BS3, bis[sulfosuccinimidyl] suberate.