Abstract
Introduction: There is controversy over the roles of loops 1 and 2 in skeletal muscle myosin. The goals of this study were to investigate the effects of cleavage of loops 1 and 2 on the amino-acid structure of the myosin and to correlate these changes with myosin function. Methods: Heavy meromyosin (HMM) was isolated from myosin purified from rabbit psoas, and treated with trypsin for periods of time ranging between 0 and 30 minutes, which caused a cleavage of loops 1 and 2. Specific cleavage of loop 1 was performed by adding actin that binds to myosin and protects loop 2, and cleavage of both loops was achieved by an ATP-free treatment. A motility essay with a frictional load was used by adding different concentrations of α-actinin (0.5 to 1.5µg/mL), and actin velocity was recorded at 30°C in 1% nitrocellulose-treated coverslips. The myosin bands were cut from sodium dodecyl-sulfate polyacrylamide gel after trypsin proteolysis and subjected to tryptic cleavage. Myosin peptides of each band were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A model was developed to interpret the results and showed that the loop 2 was damaged before loop 1 on myosin during actin motility. Results and Discussion: Sliding velocity of actin filaments decreased consistently with increasing time during trypsin treatment. The decrease was dependent on the treatment: cleavage of loops 1 and 2 decreased the velocity from 4.05μm/sec to 2.24μm/sec after 4 minutes of enzymatic treatment, and cleavage of loop 1 decreased the velocity from 3.82μm/sec to 1.22μm/sec after 2 minutes of enzymatic treatment. These results suggest that loops 1 and 2 regulate the velocity of the myosin motor and the kinetics of the actomyosin interactions.
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