01/22/2025
By Irma Silva
Candidate: Alyssa Kennedy
Date: Thursday, February 6, 2025
Time: 10 a.m. – 12 p.m.
Location: Emerging Technologies and Innovation Center (ETIC)-100L2, Perry Atrium
Committee members:
Jeffrey Moore, Professor, Biological Sciences, University of Massachusetts Lowell
Nicolai Konow, Associate Professor, Biological Sciences, University of Massachusetts Lowell
Matthew Gage, Professor, Chemistry. University of Massachusetts Lowell
Title: Evidence for Allosteric Regulation in Cardiac Thin Filament Mechanisms Through Tropomyosin Mutations.
Abstract:
Together the thin filament components-actin, tropomyosin (Tpm), and the troponin (Tn) complex-work with myosin-containing thick filaments to drive the rhythmic beating of the heart. Calcium and myosin are the principal regulators of this process by triggering shifts in Tpm position from a blocking to a permissive position on actin with regards to myosin binding, known as the steric blocking model. Disruption of these regulatory states of Tpm by mutation has been associated with cardiomyopathy. Recent structural work has predicted key electrostatic interactions between Tpm and TnI when Tpm is in the blocking position, particularly Tpm-E114. This study investigates the Tpm mutations Tpm-E114K and Tpm-E114Q, which are hypothesized to affect thin filament activation by destabilizing interactions between Tpm and TnI. We assessed the mutations’ effects on actin binding, the percentage of motile filaments, and filament speed (Vmax) over a range calcium concentration. Both mutants showed no influence on actin binding in co-sedimentation assays. In vitro motility assays revealed that Tpm-E114K and Tpm-E114Q have similar percent motile compared to WT Tpm suggesting that steric blocking for the mutant Tpm remained intact. However, Tpm-E114K showed a drastic decrease in Vmax (0.75µm/s) compared to WT (3.05µm/s), indicative of altered actomyosin kinetics despite unaltered steric blocking. Decoupling of motility speed (Vmax) from the percentage of motile filaments demonstrates that Tpm influences actomyosin kinetics and points to a potential allosteric mechanism, where structural or dynamic changes in Tpm-Tn interaction propagate to the actomyosin interface, modulating kinetic properties. These findings challenge the view that thin filament regulation is solely governed by steric blocking and highlight the critical role of allosteric mechanisms in modulating actomyosin kinetics. Parsing the distinct contributions of steric and allosteric mechanisms is crucial for unraveling the underlying molecular disruptions in thin filament regulation that contribute to cardiomyopathies.