Date of Award

Fall 2019

Document Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

St. Maurice, Martin

Second Advisor

Eddinger, Thomas

Third Advisor

Manogaran, Anita


A central debate in protein biochemistry focuses on the mechanism by which ligands contribute to conformational changes in proteins. Two primary hypotheses describe this process: the induced-fit hypothesis and the conformational ensembles hypothesis. In contrast to the induced-fit hypothesis, the conformational ensembles hypothesis states that the protein pre-exists in multiple conformational states, with ligand binding shifting the equilibrium towards a preferred conformation. The contribution of ligand binding to large-scale conformational changes is particularly relevant to multi-domain “swinging-arm” enzymes, for which pyruvate carboxylase (PC) serves as a well characterized paradigm system. PC catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. The biotin cofactor on the biotin carboxyl carrier protein (BCCP) domain is carboxylated via a MgATP-dependent reaction in the biotin carboxylase (BC) domain. The BCCP domain then travels to the carboxyltransferase (CT) domain where the carboxyl group is transferred from the biotin cofactor to pyruvate, generating oxaloacetate. There have been conflicting proposals for how ligand binding governs carrier domain translocation in PC but, to date, there have been no attempts to directly observe the positioning of the carrier domain independent of catalytic turnover. To directly observe the equilibrium positioning of the BCCP carrier domain, site-specific cross-linking was used to trap the BCCP domain in two separate orientations during translocation. This approach enabled a detailed analysis of how BCCP domain positioning responds to substrates and allosteric effectors. These studies show that carrier domain positioning is governed by conformational selection, with the carrier domain accessing intermolecular positions irrespective of the presence or identity of the ligand(s). Further, interactions between biotin and several conserved active site residues in both the BC and CT domains serve to shift the carrier domain positioning equilibrium. These studies offer the first compelling evidence that carrier domain positional equilibrium in PC is governed by conformational selection and offer new insights into the molecular level interactions contributing to these conformational changes.

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