TERTIARY STRUCTURAL-CHANGES IN THE CLEFT CONTAINING THE ATP-SENSITIVETRYPTOPHAN AND REACTIVE THIOL ARE CONSISTENT WITH PIVOTING OF THE MYOSIN HEAVY-CHAIN AT GLY699

The conformation of myosin subfragment 1 (S1) in the vicinity of the A TP sensitive tryptophan (Trp510) and the highly reactive thiol (SH1), both residing in the ''probe-binding'' cleft at the junction of the ca talytic and lever arm domains, was studied to ascertain its role in th e mechanism of energy transduction and force generation. In glycerinat ed muscle fibers in rigor, a fluorescent probe linked to SH1 detects a strained probe-binding cleft conformation following a length transien t by altering emission intensity without detectably rotating. In myosi n S1 in solution, the optical activity of Trp510 senses conformation c hange in the probe-binding cleft caused by substrate analog trapping o f S1 in various structures attainable transiently during normal energy transduction. Also in S1 in solution, the induced optical activity of a fluorescein probe linked to SH1 shows sensitivity to changing probe -binding cleft conformation caused by nucleotide binding to the S1 act ive site. The changes in the optical activity of Trp510 and SH1 bound fluorescein in response to nucleotide or nucleotide analog binding are interpreted structurally using the S1 crystallographic coordinates an d aided by a model of energy transduction that pivots at Gly699 to cha nge probe-binding cleft conformation and to displace the S1 lever arm as during force generation. The crystallographic structure of the prob e-binding cleft in S1 resembles most the nucleotide bound conformation in the native protein. A different structure, generated by pivoting a t Gly699, better resembles the native rigor conformation of the probe- binding cleft. Pivoting at Gly699 rotates probes at SH1 suggesting tha t length transients on fibers in rigor do not cause pivoting at Gly699 or reverse the power stroke.