TnI Switch Peptide Position within Cardiac Troponin as Studied by cw-Epr and DEER

Abstract

Muscle contraction is regulated by the troponin complex which is a heterotrimer protein consisting of a Ca2+ binding subunit (TnC), an inhibitory subunit (TnI) and a Tropomyosin binding subunit (TnT). Calcium binding to TnC (‘ON’ state) initiates a series of structural changes in the thin filament proteins leading to muscle contraction. In the low Ca2+ ‘OFF’ state, the TnI subunit induces muscle inhibition through its two actin binding domains (residues 133-148 and 166-210). Another region of TnI termed the ‘switch peptide’ (residues 150-161) is essential for a complete relieve of muscle inhibition in the ON state. The position of the switch peptide is vital since it affects the whereabouts of both TnI actin binding domains. In the +Ca2+ cardiac Tn core crystal structure, the switch peptide is depicted to be close to the N-lobe of TnC (Takeda et al., Nature, 2003) but no or limited knowledge is known about its position in the absence of Ca2+. We studied the proximity of the switch peptide to two domains within Tn in both the ‘ON’ and ‘OFF’ states. Two intermolecular distances from each end of the switch peptide back to the N-lobe of TnC (TnI152/TnC35, TnI152/TnC84, TnI160/TnC55) and one intramolecular distance (TnI129/TnI160) within TnI were measured with Conventional Electron Paramagnetic Resonance (cw) and Double Electron Electron Resonance (DEER) methods. In the ‘ON’ state, both the intramolecular and intermolecular distances were less than 2.5nm with narrow distance distributions indicative of restricted movement. Upon removal of Ca2+, distances increased considerably (TnI129/160 to 5nm and TnI151/TnC35 to 3.3nm) with an accompanying increase in the distance distributions suggesting a more flexible, non-bound, switch peptide.