Structure-Function Relationship of Muscle Thin Filament Regulatory Proteins: Tropomyosin and Troponin

Tropomyosin and troponin are key regulatory proteins associated with muscle thin filaments. Regulation of muscle contraction by these complexes involves precise cooperative-allosteric transitions. Several diseases are associated with changes in these transitions. Understanding the structural basis of these transitions is critical to understand the details of the regulation of muscle contraction and how they are changed in various diseases. This thesis has two aims. The first aim was to investigate tropomyosin structure function relationship by mutagenesis and various biochemical assays. I performed 7 double residues’ mutations in seven semi-repeated motifs of Tropomyosin: K6K7E, K48K49E, R90R91E, R132S133E, R167K168E, N202N203E and R244S245E. I used several different biochemical and biophysical techniques such as Stopped flow, ITC, Cosedimentation, Circular dichroism and ATPase assays to study the impact of each mutation on actin and troponin binding and thin filaments cooperativity and switching between allosteric states. These mutations had little or no effect on actin binding, troponin binding and the actomyosin ATPase inhibition at low Ca2+. However, K48K49E, R90R91E, R167K168E, N202N203E and R244S245E reduced noticeably the level of activation in the presence of Ca2+. These effects could be explained by reduced cooperativity and a stabilisation of the blocked state. The effect was most pronounced with R167K168E suggesting a critical role of this motif in tropomyosin cooperative allosteric function. K6K7E affected end-to-end interaction between adjacent tropomyosin molecules and this led to reduce the cooperativity and interaction with actin and troponin. Since the structure of the full length troponin is not yet available, the second aim of this thesis was to study troponin structure-function using nuclear magnetic resonance (NMR) spectroscopy. I was able to produce 15N labelled TnC and reconstitute it with troponin I and T and obtain an HSQC spectra. However, the quality of the spectra suggests that using the full-length troponin in NMR based structural studies is still a challenge. Alternatively, I designed a short-length troponin complex and I obtained sufficient material labelled with 15N and obtained good HSQC spectra. Thus, I laid the foundation for an exciting NMR based study of the allosteric transitions in troponin.