Assessing the Functional Role of Titin N2A-PEVK Region in Active Muscle Contraction

Abstract

Muscle contraction is an interplay of cyclic interactions between the contractile proteins myosin and actin driven by ATP turnover. Despite extensive studies and clinical significance of muscle, there are still unanswered questions about active muscle contraction especially eccentric contractions. The sliding filament theory explains many key aspects of muscle contraction but does not adequately explain short range stiffness, enhancement of force with stretch and depression of force with shortening. We aim to provide a new insight in the mechanism of muscle contraction by further elucidating the role of titin in active muscles. Recent studies have proposed two models for the role of titin in active muscles. In model one, PEVK region of titin binds to actin upon Ca+2 activation. In the second model, N2A region of titin binds to actin upon calcium influx. In this study, we are trying to understand the molecular mechanism of active muscle contraction by identifying the F-actin binding site on titin. Using an actin sedimentation assay, we have demonstrated that a recombinantly expressed truncation of mouse titin consisting of the N2A region and the first one hundred amino acids of the PEVK region binds F-actin, validating our system. We are currently working to determine whether the binding is calcium dependent and to determine the binding constants for this association. We are also testing smaller constructs of N2A and PEVK to identify the specific amino acid sequence responsible for binding. Our studies will provide a new dimension to the existing sliding filament theory and fill gaps in the molecular mechanism of active muscle contraction and movement.