PLASTICITY IN PROTEIN SEQUENCE-FUNCTION RELATIONSHIPS

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dc.contributor.advisorBeckett, Dorothyen_US
dc.contributor.authorHe, Chenluen_US
dc.contributor.departmentChemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2021-09-17T05:31:59Z
dc.date.available2021-09-17T05:31:59Z
dc.date.issued2021en_US
dc.description.abstractAllostery is defined as the functional regulation at one site in a protein by activity at a distant site. Because of the ubiquitous occurrence and diverse cellular roles of allosteric proteins, designing novel allosteric proteins is of great interest for applications in synthetic biological and disease treatment. However, the engineering of allostery is often hindered by our limited understanding of the protein sequence- function relationship, especially at residue positions that are distant from functional sites or evolutionarily nonconserved. In this dissertation, the sequence-function relationship was investigated in the Escherichia coli biotin protein ligase (BirA) system, which serves as both an essential metabolic enzyme and a transcription regulator. In its repressor function, binding to the vitamin biotin allosterically activates BirA dimerization and the resulting repression complex assembly on the biotin operator sequence. Although the allosteric regulation is conserved among bifunctional biotin protein ligases such as BirA, their sequences, even those of functional importance, are highly divergent. The in vitro characterization of BirA super repressor variants reveals that the sensitivity of transcription repression response to input biotin concentration can be altered solely through substitution-perturbed dimerization. These single amino acid substitutions are located at sites scattered throughout the protein structure including some that are distal from the BirA dimerization surface. Computational simulations indicate that the long-range effect of substitutions on dimerization results from rearrangement of a residue network that contributes to the allosteric activation in BirA. Several loops on the BirA dimerization surface were characterized for their roles in the corepressor-induced dimerization. The study of nonconserved amino acid positions spanning these surface loops reveals that a broad range of functional response in dimerization and transcription repression can be achieved by sequence variations at the nonconserved residues. Surprisingly, the substitution outcomes poorly correlate with amino acid chemistry or evolutionary frequencies, which deviates from canonical expectations based on conserved residues. Combined, these results illustrate the plastic nature of protein sequence-function relationship and provide insight into how this plasticity functions in the mechanism and evolution of allostery in BirA. Our deepened understanding of allostery in BirA and in general may facilitate the development of synthetic allosteric proteins in the future.en_US
dc.identifierhttps://doi.org/10.13016/d6zn-iasj
dc.identifier.urihttp://hdl.handle.net/1903/27790
dc.language.isoenen_US
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pqcontrolledMolecular biologyen_US
dc.subject.pqcontrolledBiophysicsen_US
dc.titlePLASTICITY IN PROTEIN SEQUENCE-FUNCTION RELATIONSHIPSen_US
dc.typeDissertationen_US

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