Structure and dynamics of cardiac troponin: a paramagnetic relaxation enhancement NMR study

Troponin is a large ~80 kDa, dynamic, heterotrimeric protein complex located on the thin filament of striated muscle, responsible for controlling the interaction of myosin with actin. The simple binding of Ca2+ to troponin initiates a series of protein conformational changes throughout the complex that then alter protein‐protein interactions in the muscle filament, leading to contraction. Despite the wealth of structural data on troponin, defining the molecular details of the conformational changes triggered by Ca2+ binding within the intact complex is still needed and experimentally remains a challenge. In this thesis, targeting of troponin with paramagnetic nitroxide spin labels enabled the techniques of Nuclear Magnetic Resonance (NMR) and Electron Paramagnetic Resonance (EPR) to be utilised to address fundamental questions about the structure and dynamics of the cardiac muscle troponin complex. Long‐range structural information was derived from the paramagnetic relaxation enhancement (PRE) of the NMR signal in the presence of the nitroxide spin label. Inter‐nitroxide spin distances are also obtained from continuous wave and pulsed EPR techniques. Together, both approaches provided us with a movie detailing the conformational interplay between key regions of the cardiac troponin complex in response to Ca2+ binding and phosphorylation. More importantly, incorporation of a paramagnetic species NMR have allowed us to understand the key role that dynamics plays in fine‐tuning the Ca2+ troponin switch regulatory mechanism in the cardiac isoform, and how its structure is perturbed by disease causing mutations.