Role of residues 311/312 in actin-tropomyosin interaction - In vitro motility study using yeast actin mutant E311A/R312A

According to the Lorenz ct al, (Lorenz, M., Poole, K. J., Popp, D., Rosenba um, G., and Holmes, K. C. (1995) J. Mol. Biol, 246, 108-119) atomic model o f the actin-tropomyosin complex, actin residue Asp-311 (Glu-311 in yeast) i s predicted to have a high binding energy contribution to actin-tropomyosin binding. Using the yeast actin mutant E311A/R312A in the in vitro motility assays, we have investigated the role of these residues in such interactio ns. Wild type (wt) yeast actin, like skeletal ar-actin, is fully regulated when complexed with tropomyosin (Tm) and troponin (Tn), Structure-function comparisons of the wt and E311A/R312A actins show no significant difference s between them, and the unregulated F-actins slide at similar speeds in the in vitro motility assay. However, in the presence of Tm and Tn, the mutati on increases both the sliding speed and the number of moving filaments at h igh pCa values, shifting the speed-pea curve nearly 0.5 pCa units to the le ft, Tm alone (no Tn) inhibits the motilities of both actins at low heavy me romyosin densities but potentiates only the motility of the mutant actin at high heavy meromyosin densities, Actin-Tm binding measurements indicate no significant difference between wt and E311A/R312A actin in Tm binding. The se results implicate allosteric effects in the regulation of actomyosin fun ction by tropomyosin.