The regulation of actomyosin by tropomyosin and troponin: Actin substructure and dynamics.

摘要

Tropomyosin (Tm) and troponin (Tn) are the main regulatory proteins responsible for the Ca2+-dependent modulation of skeletal muscle contraction. Using a variety of actin mutants and actin modifications, we investigated which actin residues interact with the regulatory complex, and actin dynamics accompanying this regulation.; Beginning in actin subdomain 3, we looked at the roles of charged residues Glu311 and Arg312 in the interaction of Tm and actin. We determined that yeast actin is a convenient model for skeletal actin in the study of regulation. We uncovered notable differences in regulation between wt and 311/312 mutant actin, with no significant differences between the two actins in their binding of Tm, S1, and S1•ADP, and small, if any differences in other non-regulated functions. Our results underscore the importance of allosteric factors in the regulation of actomyosin interactions.; We next investigated the role of subdomain 2 in regulation using labeled yeast actin mutants, labeled skeletal actin, and skeletal actin modified through controlled proteolysis. While there appear to be no discernable regulation-based subdomain 2 movements during regulation, we found that the presence of regulatory complex has an effect on the rate of ADP release, i.e., on the kinetics of the in vitro motility actomyosin cross-bridge cycle. This provides the first experimental link between the potentiation of actin motility by Tm/Tn and kinetic steps in the cross-bridge cycle. Our results also raise the possibility that direct interactions between Tm and S1 are responsible for the changes in the ADP off rate.; Finally, due to its reported role as the area of interaction for both myosin and troponin I (TnI) during the cross-bridge cycle and in regulation, we investigated the N-terminal region in actin. To study the role of these electrostatic contacts in the actin-TnI interaction, we used yeast actin mutants with both decreased and increased numbers of acidic residues in the N-terminal region. We found that the functional interaction between TnI and actin is insensitive to changes in charge at the N-terminus of actin, suggesting that other sites on actin are involved in binding the inhibitory segment of TnI.