As I attend more and more muscle and cradiac muscle talks at BPS, I realize how much the focus has moved from just studying protein function, to appreciating and studying the much more dynamic changes in proteins via structural studies and post-translational modifications like phosphorylation, methylation, oxidation and ubiquitination. Exchange studies wherein phosphomimetics prepared by single site directed mutagenesis are created and exchanged within the muscle preparations to study these effects. PTM (post-translational modifications) sites are identified using 2-D DIGE studies, mass spectrometry, IEF gels, ProQ diamond staining, phospho-tags.
The mechanics and kinetics of cross-bridge cycling are then studied by langendorff hearts, intact fiber preparations, single cell studies, single myofibril technique, in-vitro motlity assays, 3- bead assay and exchange studies. Mechanical data are extensively being employed to study the nature of these PTM exchanges to understand how they regulate muscle function. Structural studies using X-ray diifraction, NMR, circular dichroism and mass spectrometry are key tools to really look at the molecular structure and protein folding to understand these phosphomimetics to see what changes within the molecule/s.