Unraveling the detailed molecular mechanisms involved in muscle contraction is a prerequisite for understanding the molecular basis of muscular disorders. Troponin (Tn) plays a key role in muscle regulation, and it is composed of three subunits: TnC, TnI and TnT. This study is aimed at understanding conformational changes occurring in TnI during muscle activation and force generation. The primary experimental technique used is site specific spin labeling combined with electron paramagnetic resonance (SDSL-EPR).
In present study, we used SDSL EPR technique to observe the orientational changes of TnI-TnT coiled-coil domain and the conformational changes of TnI C-terminal domain between different thin filament states. The fiber tilts spectra of TnI133-MTSSL reconstituted fibers showed that in this domain, TnI is well ordered and oriented at a specific angle with respect to the actin filament. After simulations using a nonlinear-least-squares (NLS) approach, we found that TnI-TnT coiled-coil undergoes a significant orientational change between different states: from “Blocked” to “Closed” state, TnI re-orients itself ~50° axially; from “Closed” to “Open” state, TnI re-orients itself 90° azimuthally. In order to examine the conformational change of TnI C-terminal region upon Ca2+ binding, DEER distance measurements between two residues in TnI C-terminal region (residue 170 and residue 202) were performed. The broad distance distribution between residue 170 and 202 indicates that the TnI C-terminal region is flexible. Unlike TnC and other regions of TnI, in solution, this region is not affected by Ca2+ binding. The reconstitution of the troponin complex to Tm/actin does not change the flexibility of the TnI C-terminal region. By using both DEER and FRET techniques to monitor the distance change between TnI and actin upon Ca2+ binding, we discovered that this region shifts away from actin in thin filaments when Ca2+ is bound. This is the first direct observation of such a conformational change.
