Calcium-Regulated Conformational Changes in the COOH-terminus of Troponin I

Abstract

The troponin complex plays an essential role in the calcium regulation of skeletal and cardiac muscle contractions. Of the three subunits of troponin (TnC, TnI and TnT), TnI is the inhibitory subunit that responds to the binding of Ca2+ to TnC during the activation of contraction. The COOH-terminal region of TnI is a highly conserved structure implying a fundamental function. Previous studies using reconstituted troponin or myofilaments suggested that the COOH-terminal domain of TnI undergoes epitopic and positional changes in the presence or absence of calcium. Here we tested the calcium-induced conformational changes in the COOH-terminal region of TnI by engineering a unique Cys at the COOH terminus of TnI for the addition of a reporting label. Monoclonal antibody epitope analysis and protein binding assays indicated that this modification and the replacement of two internal Cys residues (C81I and C98S) in a cardiac TnI core structure (McTnI-ND29-Cys) did not affect the COOH-terminal conformation of TnI and preserved binding to TnT and TnC. McTnI-ND29-Cys purified from bacterial culture was fluorescently labeled with the Alexa Fluor 532 dye and used to reconstitute troponin complex. After verifying the ratio of fluorophore to protein conjugation by spectrophotometer and SDS-PAGE, Ca2+-titrations were performed for fluorescence intensity and polarization changes. The results demonstrated Ca2+ regulated conformational/environmental changes as well as flexibility change in the COOH terminus of TnI. Further experiments are performed to measure the Ca2+-induced structural changes in reconstituted myofilaments to understand the function of TnI COOH terminal domain in calcium-regulation of muscle contraction.