Doctoral Dissertations

Orcid ID

Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Life Sciences

Major Professor

Dean A. A. Myles

Committee Members

Barry D. Bruce, Dean A. A. Myles, Hugh M. O'Neill, Jerry M. Parks, Matthew J. Cuneo


Substrate binding proteins (SBPs) are ubiquitous in all life forms and have evolved to perform diverse physiological functions, such as in membrane transport, gene regulation, neurotransmission, and quorum sensing. It is quite astounding to observe such functional diversity among the SBPs even when they are restricted by their fold space. Therefore, the SBPs are an excellent set of proteins that can reveal how proteins evolution novel function in a structurally conserved/constrained fold. This study attempts to understand the phenomenon of affinity and specificity evolution in SBPs by combining a set of biochemical, biophysical, and structural studies on the SBPs involved in translocation of substrates across the membrane using ATP-binding cassette (ABC) transporters and tripartite ATP-independent periplasmic (TRAP) transporters in gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa, respectively. Additionally, experimental, and computational methods were used in conjunction to highlight the variation in the dynamics of these SBPs. The results from this study highlight an intricate role of dynamics in complementing the structural alterations that are required for high-affinity ligand binding. Moreover, first ever neutron structure of a SBP was determined during my study to delineate the extensive network of water in the binding cavities of the SBPs that help stabilize larger substrates by forming water-mediated hydrogen bond interactions with the bound substrates. Furthermore, structures of two SBPs from T. maritima were determined in both substrate-free (apo) and substrate-bound (holo) forms and subsequently used for computational molecular dynamics simulation to determine the variation in dynamics due to substrate-binding. The novel TRAP SBP identified in P. aeruginosa was identified as a promiscuous binder of several tricarboxylic acid cycle (TCA) cycle intermediates. A total of six SBP structures were determined using X-ray crystallography and one SBP structure was determined using neutron crystallography. Finally, experimental neutron scattering was used to experimentally characterize the picosecond to nanosecond dynamics in SBPs and highlighted differences in the translational, rotational, and internal dynamical signatures of two SBP isoforms. Overall, the findings of this study can be broadly applied in biotechnology and biosensor development by artificially engineer affinity or specificity for a particular ligand.

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