Structural Dynamics of the C-Domain of Cardiac Troponin I In the Reconstituted Thin Filament

Abstract

The inhibitory region (Ir), regulatory region (Rr), and mobile domain (Md) within the C-domain of cardiac troponin I (cTnI) play different functional roles in regulating the thin filament switch. The functional role of each region is believed to be associated with structural dynamics of the region. This study used fluorescence polarization to acquire dynamic information on the C-domain of cTnI in the reconstituted thin filament. To achieve this goal, a series of cTnI mutants were generated with single-cysteine at residues 131, 145, and 151 within the Ir, residues 160 and 167 within the Rr and residues 177, 182, 188, 200, and 210 within the Md. In addition, double-cysteine cTnI(177C/188C) and cTnI(182C/200C) were also generated to investigate the conformational change of the Md in response to Ca2+ regulation using homo-FRET. These cTnI mutants were labeled with fluorophore TAMRA and reconstituted with other proteins to form thin filament. The reconstituted samples were subjected to steady-state and time-resolved fluorescence anisotropy measurements, and fluorescence quenching experiments under different biochemical conditions. The anisotropy measurements showed that the Ir was less dynamic than the Rr and Md at both Mg2+ and Ca2+ states. Ca2+ binding to troponin induced minimal changes in Ir dynamics, but significantly decreased Rr flexibility, and resulted to a more compact Md with more dynamics as whole. Furthermore, Cys210 at the C-terminus of cTnI was highly immobilized comparing to other segments of the domain in the Mg2+ state, and became highly dynamic in the Ca2+ state, suggesting a unique role of the C-terminus in regulating thin filament switching. Fluorescence quenching results, the effects of the strongly-bound S1 on the structural dynamics of the C-domain of cTnI, and a detailed data analysis will be discussed in this presentation.