Browsing by Author "Nguyen, Peter Q."

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    Effect of protein thermostability on the cooperative function of split enzymes
    (2010) Nguyen, Peter Q.; Silberg, Jonathan J.
    Although 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.
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    Protein fragment complementation assay for thermophiles
    (2012-08-07) Silberg, Jonathan; Nguyen, Peter Q.; Rice University; United States Patent and Trademark Office
    A protein fragment complementation assay for thermophiles is provided wherein a thermophilic bacteria having a temperature-sensitive adenylate kinase is transformed with one or more vectors having sequences encoding a first test peptide operatively fused to a first portion of a thermostable adenylate and a second test peptide operatively fused to a second portion of the thermostable adenylate kinase. Association of the first and second test peptides allows association of the first and second portions of the thermostable adenylate kinase and growth of the thermophilic bacteria at a temperature greater than 70° C.
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    Random Insertion of mCherry Into VP3 Domain of Adeno- associated Virus Yields Fluorescent Capsids With no Loss of Infectivity
    (American Society of Gene & Cell Therapy, 2012) Judd, Justin; Wei, Fang; Nguyen, Peter Q.; Tartaglia, Lawrence J.; Agbandje-McKenna, Mavis; Silberg, Jonathan J.; Suh, Junghae
    Adeno-associated virus (AAV)-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase). The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.