Troponin I Inhibitory Region’s Role in Cardiac Muscle Regulation and Troponin Structural Dynamics

To maintain cardiovascular homeostasis, the heart adapts to circulatory needs and/or stress in a greatly malleable process involving specific regulatory mechanisms. Troponin plays a crucial role in such regulation, by acting as the protein on-off switch of the cardiac muscle contractile apparatus. Within troponin, troponin I (TnI) is considered to be the critical troponin subunit for switching off the heart in the absence of Ca2+, in significant part via the action of a small portion of TnI termed the inhibitory peptide region. This thesis examines the role of the inhibitory region of TnI in cardiac muscle regulation, as well as the effects of the inhibitory region on overall troponin dynamics both locally and at a distance. The first part of this thesis explains how troponin's inhibitory effects have previously been shown to be mimicked by this 12-residue TnI peptide, termed the inhibitory peptide. Mutagenesis of this segment within the whole troponin context can be used to investigate how the inhibitory peptide region alters the thin filament to affect or effect regulation. Accordingly, we describe the functional properties of troponins containing mutations within the inhibitory peptide sequence. Smaller or larger portions were replaced with Gly-Ala linkers. The results show that the mutations impaired Ca2+-mediated regulation of both ATPase rates and myosin binding to the thin filament. The regulatory impairment was considerable, supporting an important role for the inhibitory peptide region. On the other hand, even for thin filaments with complete replacement of the inhibitory peptide region, Ca2+ addition caused a cooperative increase in myosin-thin filament ATPase activity. This points to the co-importance of other parts of troponin as mediators of inhibition and activation generally, and of cooperative regulation specifically. The relationship of these findings to the overall regulatory mechanism is discussed. In the second part of the thesis, we use hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) to study the dynamic properties of troponin composed of cTnT, cTnI with the complete inhibitory region replaced with a flexible linker and cTnC with mutational inactivation of site II Ca2+ binding. Additionally, we study the dynamics of a troponin complex similar to the one above but with wild type TnC instead. This allowed the determination of the effects of the inhibitory region on troponin dynamics both in the presence and absence of Ca2+. HDX allows for the mapping, in detail, quantitatively, of the dynamic properties of each part of cardiac troponin. H-D exchange rates in folded proteins tend to track with local folding instability. Therefore, measurements of local rates of exchange across a protein provide quantitative dynamic information on various distinct regions of that protein. We propose that, in the absence of Ca2+ bound to regulatory site II of TnC, the Ip region has unexpected importance for stabilizing intra-troponin interactions relatively distant from the Ip region itself. Partial explanation may come from a mechanism involving the inhibitory peptide being highly flexible, and this flexibility being important for critical changes in troponin structure. In presence of Ca2+ bound to regulatory site II of TnC, the mutational alteration of troponin dynamics is less than in the absence of Ca2+, but occurs much more widely than is directly predictable from the high resolution structure of troponin. Collectively, the results shed new insight onto the role of troponin’s inhibitory region as well as the broad mechanism of troponin’s regulatory function.