The long-term goal of this project is to provide a better understanding of the interconnections between internal dynamics, protein structures, and functions. The internal dynamics of backbone and sidechain atoms of wild-type and functionally altered mutants of the 25 kDa tryptophan repressor (TrpR) protein will be studied using NMR relaxation approaches. TrpR is part of a large family of bacterial proteins that regulate the expression of metabolic genes by binding to DNA operator sequences in response to cellular needs. The repressor's affinity for DNA is controlled by L-tryptophan (L-trp), an allosteric effector that activates the TrpR. The intrinsic flexibility of the TrpR protein structure is thought to be at the origin of the non-cooperativity of binding of the L-tryptophan corepressor to TrpR, and of the non-local long-range effects observed in a temperature-sensitive mutant of the tryptophan repressor protein, L75F-TrpR, which cannot be simply explained by small structural changes when compared to the wild-type TrpR. A second TrpR mutant of interest is A77V which, like L75F, is structurally similar to wild type TrpR and possesses biophysical features analogous to those of L75F-TrpR. Despite similar structures and biophysical features, the two TrpR mutants yield very distinct phenotypes and have very different L-trp cofactor binding properties. Such biochemical differences cannot be explained by the presence of distinct structural changes. The hypothesis to be examined with the NMR relaxation experiments is that the source of the differential L-trp binding properties of the A77V and L775F TrpR proteins originate from differences in intrinsic flexibility and molecular mobility which cannot be identified from inspection of the protein structures. The objective of this project is to provide new insights into the biochemical role of internal motions in modulating TrpR-L-trp-cofactor complex formation and TrpR-DNA recognition.
This research will train graduate and undergraduate students in cross-disciplinary research involving NMR-based structural biology and fundamentals of protein chemistry. In addition, the PI will use knowledge obtained from this research to develop a graduate level course in biophysics focusing on the thermodynamics of ligand binding, protein-protein and protein-nucleic acid interactions, role of structural stability and dynamics in modulating protein functions, and allosteric regulations of multivalent protein systems.
