Faculty Opinions recommendation of Disease-associated mutant alpha-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity.

ResultsWe initiated our studies by investigating the interaction of ␣-actinin-4 with actin in vitro by using purified recombinant K255E mutant and WT ␣-actinin-4 proteins.After confirming that K255E mutant ␣-actinin-4 alone does not aggregate in vitro [supporting information (SI) Fig. 7], we performed a detailed actin-binding analysis by using a standard actin cosedimentation assay.Representative Coomassie-stained polyacrylamide gels are shown in Fig. 1a.As shown in Fig. 1 b and c, the dissociation constant (K d ) of K255E ␣-actinin-4 was 0.046 M, almost 6-fold lower than that of WT ␣-actinin-4 (0.267 M).The K255E ␣-actinin-4 dimers saturate F-actin with a 1:2 stoichiometry, twice the ratio of WT dimers (1:4).