Comparative studies of protein structure and dynamics by molecular dynamics simulation, NMR spectroscopy and x-ray crystallography

grantor: University of TorontoIn recent years, Molecular Dynamics (MD) simulations have been used successfully along with experimental methods for the joint investigation of the biophysical and biological properties of protein molecules. Here, the results of MD simulations of three protein systems are reported, in conjunction with, mainly, data from Nuclear Magnetic Resonance (NMR) spectroscopy and X-ray diffraction. These three techniques are outlined in Chapter 1. The systems under study are: 'Bacillus stearothermophilus' L-lactate dehydrogenase (BSLDH), the C-terminal SH2 domain of phospholipase C- g 1 (PLCC), and 'E. coli' ribonuclease HI (RNase HI). A high-temperature MD simulation of BSLDH is used to investigate the mechanism of closure of a catalytically crucial loop, as this phenomenon is thought to be rate-limiting and essential for catalysis (Chapter 2). The phosphotyrosine-binding region of the PLCC domain is thought to account for most of the binding free energy of this domain complexed with high-affinity phosphopeptides. Consequently, three MD simulations of PLCC, in its free form and bound to either phosphate ion or phosphopeptide, are performed to elucidate interatomic interactions in this region and interpret experimental chemical shift data (Chapter 3). A comparison of an MD trajectory of RNase HI with NMR generalised order parameters of backbone amide bonds and crystallographic thermal factors is presented (Chapter 4). The relative accuracy and precision of MD and NMR amide order parameters are assessed through comparisons among three NMR and two MD independent data sets (Chapter 5). Furthermore, an ensemble of 15 NMR structures of the protein is searched for dynamic information through correlation with data from two crystal structures and the MD simulation (Chapter 6). The work presented herein demonstrates the value of the combined use of theoretical and experimental methods for studying protein structure, dynamics and function (Chapter 7).Ph.D