FIGURE 5.

Sm-titin Zq domain binding to wild type and mutated smooth muscle α-actinin R2-R3 domains in vitro. A–C, wild type and mutant smooth muscle α-actinin R2-R3 domains expressed as GST fusion proteins and immobilized on glutathione beads were incubated with an expressed wild type smooth muscle Zq domain fragment in buffer containing 100 mm KCl and 0.2% Triton X-100. T, titin; MHC, myosin heavy chain. A, SDS-PAGE gel from a representative experiment shows unbound Zq domain in the supernatant and in washes (lanes 2–4), Zq domain that bound to the α-actinin R2-R3 domain on the beads and coeluted with SDS (lane 5), and R2-R3 fragment alone bound to and eluted from the beads (lane 6). Lane 1 contains a chicken pectoralis muscle extract used as a molecular weight standard. B, a control experiment demonstrates that the Zq domain fails to bind to beads lacking the α-actinin R2-R3 domain (lane 4). C, the Zq region of a gel showing the amount of bound Zq (equivalent to A, lane 5) from experiments in which R2-R3 domains with the specified mutations were bound to the beads reveals variable effects of the mutations on Zq binding. The mutations tested include the R2 loop mutations K421A, Y423A, and T425A, the R3 loop mutations N586A, T590A, N591A, and the phosphomimetic mutation T427D of a putative CK2 phosphorylation site. The signs indicate the lanes in which Zq binding was detectable (+) or undetectable (-) on the gel.