Biological Structure and Dynamics at the Interface of Experiment and Theory

The research presented in this thesis reflects advances in the adaptation and application of NMR crystallography – the synergistic combination of X-ray crystallography, NMR spectroscopy, and computational methods – to the active sites of enzymes. The goal is to construct highly detailed and chemically rich structures of the enzyme active site. The direct targets of this work are kinetically competent, quasi-stable intermediates along the reaction pathway of the pyridoxal-5’-phosphate dependent enzyme tryptophan synthase. NMR crystallography relies on the availability of both X-ray crystal structures and NMR data. This study makes use of multiple NMR restraints, including13C, 15N, and 31P NMR chemical shifts measured by collaborators in the Mueller group, and adds to these by including 17O as an active site reporter. The acid-base catalysis featured in many enzyme mechanisms relies on oxygen as a key atomic species in amphiprotic functional groups, making it a potentially important probe nucleus for NMR crystallography in enzyme active sites. Yet oxygen is not considered a standard biological NMR probe given the perceived difficulties related to its quadrupolar nuclear status. In this work we utilize solution state 17O Quadrupole Central Transition NMR to directly measure 17O chemical shifts and probe ionization states of enzyme bound intermediates.The NMR shifts are combined with X-ray crystallography to build computational models of the active site consistent with both the NMR and X-ray data. This thesis provides an exposition of the synergy of the NMR crystallographic approach applied to important intermediates of tryptophan synthase’s catalytic cycle. The results of this approach demonstrate the unprecedented level of structural detail that can be revealed, most remarkably for proton locations, within the active site. This is highlighted in particular by the characterization of proton mediated tautomerization in the “quinonoid/carbanion” intermediate, where first principle calculations of charge allow the mechanistic implications of the observed protonation states to be fully appreciated