A Novel Tryptophan Analog Designed for Studying Calcium Induced Conformational Change of Troponin C

Abstract

Cardiac troponin C (cTnC) is an integral component of the trimeric cardiac troponin (cTn) complex that binds periodically to every 7th actin monomer and tropomyosin to make up the thin filament. Binding of Ca2+ to cTnC induces a conformational change that results in the cascade of events leading to muscle contraction. To date over seventy cTn mutations have been found associated with familial hypertrophic cardiomyopathy (FHC), which is the leading cause of sudden cardiac death in young individuals. Altered Ca2+ handling has been shown to be one of the major underlying causes of the FHC-induced arrhythmogensis. Ca2+ binding to TnC induces a protein conformational change, which can be recorded by the change in fluorescence of appropriately placed fluorophores. The commonly used fluorophore IAANS is bulky and requires at least one Cys to Ser mutation to allow attachment to naturally occurring or an engineered Cys. However, such a mutation in the highly conserved cTnC is not completely innocuous in terms of structure and function of the protein. For this purpose, we have developed a novel boron-dipyrromethane (BODIPY) containing tryptophan analog with specific spectral properties. It is optimized with an excitation wavelength of 490 nm and with a peak of emission at 530 nm. This fluorophore is incorporated at residue 27 of cTnC. We have successfully expressed and incorporated this tryptophan-analog using a minimal media expression system and an auxotrophic E. coli cell strain (CY(DE3)). Preliminary experiments with this tryptophan analog have confirmed its ability to report conformational change of isolated TnC upon Ca2+ binding. Experiments that report Ca2+ binding to cTnC in the cTn complex, as well as in a more physiologically relevant biochemical system, the reconstituted thin filament, are underway.