orals 5th Asia-Pacific NMR Symposium 2013

Using the complementary approaches of EPR and NMR to construct a dynamic picture of Troponin - the calcium switch in muscle (#73)

Louise J Brown 1 , Nicole M Cordina 1
  1. Macquarie University, North Ryde, NSW, Australia

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.

We have used both EPR and NMR in the presence of a paramagnetic nitroxide spin label to describe the structure and dynamics of the cardiac troponin muscle complex. Site directed spin labeling (SDSL) of troponin is achieved through cysteine mutagenesis and the covalent attachment of the paramagnetic nitroxide spin label. It is ideally suited for probing troponin through all its levels of structural complexity, from isolated subunits to whole reconstituted muscle filaments. Long-range structural information is 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 have 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, the complementary approaches of EPR and 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 mutations1,2.

  1. Cordina, Liew, Gell, Fajer, Mackay, Brown. Protein Science, 21: 1376 (2012)
  2. Cordina, Liew, Gell, Fajer, Mackay, Brown. Biochemistry 52:1950 (2013)