β-cardiac myosin subfragment 1 (βS1) tertiary structure and dynamics were characterized with proteolytic digestion, nucleotide analogue trapping kinetics, and intrinsic fluorescence changes accompanying nucleotide binding. Proteolysis of βS1 produces the 25, 50, and 20 kDa fragments and a new cut within the 50-kDa fragment at Arg369. F-actin inhibits cleavage of the 50-kDa fragment and fails to inhibit cleavage at the 50/20 kDa junction, suggesting βS1 presents an actoS1 conformation fundamentally different from skeletal S1. Time-dependent changes in Mg2+-ATPase accompanying proteolysis identifies cleavage points that lie within the energy transduction pathway. The nucleotide analogue trapping kinetics reveal the presence of a reversible weakly actin attached state. Comparison of nucleotide analogue induced βS1 structures with the transient structures occurring during ATPase indicates analogue induced and transient structures are in a one-to-one correspondence. Tryptophan fluorescence enhancement accompanies the binding or trapping of nucleotide or nucleotide analogues. Isolation of Trp508 fluorescence shows it is an ATP-sensitive tryptophan and that its vicinity changes conformation sequentially with the transient intermediates accompanying ATPase. These studies elucidate energy transduction and suggest how mutations of βS1 implicated in disease might undermine function, stability, or efficiency.
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