Characterization of Multiple Surface Loops in Escherichia coli Biotin Protein Ligase Functions

Simple item page
dc.contributor.advisorBeckett, Dorothyen_US
dc.contributor.authorAdikaram, Poorni Ranmalien_US
dc.contributor.departmentBiochemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2013-02-08T06:32:17Z
dc.date.available2013-02-08T06:32:17Z
dc.date.issued2012en_US
dc.description.abstractMultifunctional proteins utilize several strategies to interact with different partners, resulting in diverse cellular outcomes. Structural, thermodynamic and kinetic features of these proteins influence the switch between functions. The Escherichia coli biotin protein ligase, BirA, is a bifunctional protein essential for biotin homeostasis. It transfers biotin to the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase in its metabolic role or dimerizes and binds the biotin biosynthetic operator as a transcriptional repressor. Each function involves forming a protein-protein interaction, and because a single surface of BirA is used to form both interactions, the two are mutually exclusive. The BirA interaction surface contains several loops, two with highly conserved sequences, and the remaining with variable sequences. In this work the roles of four loops in facilitating BirA function were investigated. Amino acids from surface loops were replaced with alanine to obtain 18 alanine substituted variants. Homodimerization energetics measured using sedimentation equilibrium yielded an 8 kcal/mol range for variants from all loops. Steady-state and stopped-flow kinetic assays yielded 7 of 18 variants that exhibited slower rates than wild-type in biotin transfer to BCCP. The majority of alanine substituted variants are from constant loops. These results indicate that the biotin transfer reaction is mediated primarily through the constant loop and homodimerization is facilitated by all surface loops. The energetics of transcription repression complex assembly, which comprises contributions from dimerization and DNA binding, was assessed using DNaseI footprint titrations. Although variants exhibit a broad range in total assembly energetics, all dimers bind with similar affinities to DNA, implying independence between DNA binding and dimerization domains. The switch between functions was also investigated using inhibition DNaseI footprint titrations. A direct correlation between inhibition of repression complex assembly and rates of BirA-BCCP association was observed, reinforcing a kinetic mechanism for the switch between BirA functions. These studies indicate that multiple surface loops form the structural basis for bifunctionality, and BirA switches between protein-protein interactions through a kinetically controlled mechanism. Elucidation of structural and mechanistic aspects of the BirA functional switch enhances our understanding of how multifunctionality evolves and the mechanism of switching between biological functions.en_US
dc.identifier.urihttp://hdl.handle.net/1903/13663
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pquncontrolledevolutionen_US
dc.subject.pquncontrolledfunctional switching mechanismsen_US
dc.subject.pquncontrolledProtein-protein interactionsen_US
dc.subject.pquncontrolledsurface loopsen_US
dc.titleCharacterization of Multiple Surface Loops in Escherichia coli Biotin Protein Ligase Functionsen_US
dc.typeDissertationen_US

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Adikaram_umd_0117E_13748.pdf
Size:
2.8 MB
Format:
Adobe Portable Document Format
Download

Collections

UMD Theses and Dissertations
Chemistry & Biochemistry Theses and Dissertations