FIGURE 4:

Examination of the effects of the Y247H and E248K mutations on the ability of the NH₂ terminus of sMyBP-C to bind actin and myosin in vitro. (A) Coomassie blue–stained gel showing equivalent amounts (1.5μg) of control glutathione S-transferase (GST), wild-type GST-P/A-C1-M, or mutant GST-P/A-C1-M proteins used in the overlay assays. Full-length GST-P/A-C1-M recombinant proteins (~58kDa) are denoted with an arrowhead; a degradation product at ~50kDa is present in all three protein preparations. (B) Equivalent amounts (3μg) of purified heavy meromyosin (HMM) or actin were separated by SDS-PAGE, transferred to nitrocellulose membrane, and overlaid with 0.5μg/ml of control GST protein, wild-type GST-P/A-C1-M, or mutant GST-P/A-C1-M carrying the Y247H or E248K mutation. Nitrocellulose membranes (Ponceau stain) and films (western blots probed with GST-ab) were cropped to only include the area of expected signal. (C) Quantification of immunoblots following the overlay assays indicated that both mutant proteins exhibit significantly increased binding to HMM (~2-fold for Y247H and ~3.5-fold for E248K) compared to wild-type protein (n = at least three independent repeats; t test; *p < 0.04 and ^#p < 0.015, respectively); control GST protein did not bind to either myosin or actin. GST-ab = glutathione S-transferase antibody; SDS-PAGE = sodium dodecyl sulfate/polyacrylamide gel electrophoresis.