Actin is a major component of thin filaments in sarcomeres. Tropomyosin (Tpm), a coiled-coil protein, polymerizes in a head-to-tail manner and binds along the sides of actin filaments, stabilizing the filaments. At the pointed filament ends, it interacts with tropomodulin (Tmod) and leiomodin (Lmod) maintain the uniform length of thin filaments. In addition to its role in maintaining thin filament stability, Tpm interacts with troponin (Tn) to regulate calcium-dependent muscle contraction. Two cardiomyopathy-linked mutations have recently been identified in a cardiac isoform of Tpm. These mutations, K15N and R21H, are associated with dilated and hypertrophic cardiomyopathies, respectively. However, the molecular mechanisms underlying their clinical consequences are not understood. I hypothesize that (a) these mutations destabilize Tpm's coiled-coil structure and consequently reduce Tpm's affinity for its binding partners, and (b) the mutations affect the overall conformation and calcium-dependent dynamics of thin filaments in opposite manners, underlying development of cardiomyopathies causing opposite phenotypic responses of the heart. The hypothesis will be tested in two specific aims. In Aim 1, effects of the mutations on Tpm's coiled-coil conformation and affinity for F-actin, Tmod, Lmod and Tn will be studied using circular dichroism, molecular dynamics simulation, co-sedimentation, and pyrene-actin polymerization assay. In Aim 2, Frster resonance energy transfer (FRET) will be used to examine how the Tpm mutants affect calcium sensitivity, calcium binding cooperativity, and deactivation kinetics of thin filaments. FRET will also be used to understand how the mutations induce structural changes within the troponin complex. By discovering the molecular links between the mutations and their clinical consequences, this study will facilitate early and effective molecular diagnosis and development of drugs and therapies for these heart diseases.
|Program type||Predoctoral Fellowship|
|Effective start/end date||07/01/2017 → 11/05/2018|