This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The ribosome is an important molecular machine in living cells in which proteins are synthesized via peptide bond formation (PBF) between amino acid units. PBF is an aminolysis of the esteric bond of the peptidyl-transfer-RNA by the amino group of the aminoacyl-transfer-RNA. The detailed chemical mechanism of PBF in the ribosome, despite numerous and valuable insights from crystallographic, kinetic, and computational studies, has yet to be fully elucidated, including the specific roles played by the ribosomal environment, i.e. how some key RNA groups and the few waters present in the peptidyl transferase center catalyze PBF. Because of both the rather large size of the relevant ribosomal environment and the significant number of atoms directly involved in the reactive event that need to be treated quantum chemically, QM/MM technology is required. We will adopt a two-step approach to the study of the PBF chemical mechanism: (a) Transition state optimization of the core reaction system (CRS) --- comprising the atoms most directly involved in the reaction --- embedded in the ribosomal environment, followed by the calculation of the intrinsic reaction coordinate path (IRCP) from reactants to products --- the CRS will be treated ab initio at the DFT/B3LYP/6-31G* with the surrounding environment described by the classical AMBER force field. (b) QM/MM MD simulations using umbrella sampling in several assumed reaction coordinates suggested by the previous step to calculate the reaction free energy --- the simulations will use the CHARMM and Q-CHEM software, or some other packages suitable for QM/MM simulations.
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