Structural Transitions within the Actomyosin Complex in the Presence of Saturating ATP, Detected by Time-Resolved FRET

Abstract

Myosin generates force by cycling between actin-attached (strong binding) and actin-detached (weak binding) states during the actomyosin ATPase cycle. However, it remains unclear what specific conformational changes in myosin take place upon binding to actin, and how these structural changes lead to product release and force. To characterize these changes, we have used site-directed FRET to determine distance between specific sites in actin and myosin subfragment 1 (S1) in the presence and absence of ATP. Rabbit skeletal muscle myosin has two essential light chain (ELC) isoforms, A1 and A2.The ELC isoform has significant effects on the interaction of S1 with actin, resulting in higher catalytic efficiency for the S1A1-isoenzyme but higher in vitro motility and muscle shortening velocity for S1A2.We have focused on S1A1, since it binds to actin in the presence of ATP with higher affinity. Actin was labeled at C374 with a donor, AlexaFluor 568. A1 light chain, was expressed,purified and labeled at C177 with an acceptor, AlexaFluor 647. The labeled A1 was exchanged into myosin S1, and S1A1 was isolated using a Talon affinity resin. The efficiency of energy transfer between C374 of actin and C177 of A1 was measured by time-resolved fluorescence, allowing us to detect directly the FRET within the actin-S1A1 complex, even in the presence of saturating ATP. Addition of ATP to the strongly bound complex resulted in a significant decrease in FRET within the weakly bound complex, corresponding to a significant increase in the donor-acceptor distance. We conclude that the transition between strongly and weakly bound states of acto-S1 is associated with a structural transition in the acto-S1 complex that affects the distance from actin to the light-chain domain of myosin.