Effect of protein thermostability on the cooperative function of split enzymes

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dc.contributor.advisorSilberg, Jonathan J.en_US
dc.creatorNguyen, Peter Q.en_US
dc.date.accessioned2011-07-25T01:51:01Zen_US
dc.date.available2011-07-25T01:51:01Zen_US
dc.date.issued2010en_US
dc.description.abstractAlthough the effects of mutational events on protein function cannot yet be predicted a priori, molecular evolution studies have shown that the tolerance of protein structure and function to random mutation is positively correlated with the thermostability of the protein mutated. To test whether thermostability also influences protein function upon random fission, I have characterized the function of split Bacillus subtilis and Thermotoga neapolitana adenylate kinases (AK Bs and AKTn, respectively), enzymes that are required to maintain adenine energy charge. Using libraries of split AKBs and AKTn variants, I show that mesophilic and thermophilic AK orthologs can be split at multiple sites into fragments that complement the growth of Escherichia coli with a temperature-sensitive AK at 40°C. However, I find that the fraction of split AKTn variants that function is ∼7-fold higher than that observed for split AKBs variants. I also find that AKTn can be split within the AMP-binding and LID domains to create functional variants, whereas AKBs can only be split within the AMP-binding domain. Biochemical and biophysical analysis of one pair of homologous split AK variants reveal that polypeptide fragments derived from the more thermostable AK exhibit greater secondary structure and enzymatic activity, suggesting that residual structure of these fragments could account for their retention in function. In addition, complementation studies show that the association and cooperative function of AKBs fragments with little residual structure can be increased by fusing these peptides to interacting proteins. Similarly, the interaction of a split AK Tn can be enhanced at a temperature (78°C) where the fragments are non-functional by fusion to proteins that interact. This split AKTn, which represents the first high-temperature protein fragment complementation assay (ht-PCA) for analyzing protein-protein interactions within a living thermophilic bacterium, is capable of detecting predicted interactions among Thermotoga maritima chemotaxis proteins. These findings show that split proteins with varying functions can be rapidly discovered by fragmenting orthologs with a range of thermostabilities. Moreover, the novel ht-PCA described herein will aid in creating genome-wide maps of thermophilic protein-protein interactions, studying the effects of temperature on biomolecular interactions, and engineering oligomeric thermostable nanomaterials.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoTHESIS BIOCHEM. 2010 NGUYENen_US
dc.identifier.citationNguyen, Peter Q.. "Effect of protein thermostability on the cooperative function of split enzymes." (2010) Diss., Rice University. <a href="https://hdl.handle.net/1911/61965">https://hdl.handle.net/1911/61965</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/61965en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectChemistryen_US
dc.subjectBiochemistryen_US
dc.titleEffect of protein thermostability on the cooperative function of split enzymesen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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