Troponin Structure And Effects Of Phosphorylation And Mutations Studied By Molecular Dynamics Simulations

The crystal structure of cTn does not include the crucial N-terminus cTnI region. We used all-atom Molecular Dynamics to understand the molecular events that follow phosphorylation in silico. We performed multiple microsecond MD simulations of wild type (WT) cTn (7250 ns so far) on a 419 amino acid cTn model containing human sequence cTnC (1-161), cTnI (1-172) and cTnT (216-298) including sequences not in the crystal structure. We have compared our results to previous computational studies, and proven that longer simulations and a water box of at least 25A are needed to sample the interesting conformational shifts both in the native and phosphorylated states. As a consequence of the introduction into the model of the C-terminus of cTnT that was missing in previous studies TnC-TnI interactions that are responsible for the cTn dynamics are altered. We used cluster analysis to select 4 of our conformations at 750ns to start simulation of bisphosphorylated (TnI Ser23/24) cTn and have collected multiple 750ns simulations totaling 2650 νs so far. We show that phosphorylation does not have an appreciable effect on the cTn fluctuations and its flexible regions. The same molecular contacts between TnI 1-30, TnI 164-168 and TnC 1-88 and between TnI 1-40 and 138-147 and between TnC 1-41 and TnT 280-298 chare sampled in both phosphorylated and unphosphorylated simulations but phosphorylation changes the locations of interactions between the cTn subunits. We will describe ongoing simulations of Ca2+-cTn with HCM mutations TnI R145G and TnT K280N and DCM mutations TnI K36Q and TnC G159D in native and phosphorylated states.