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dc.contributor.authorPuthenveetil, Sujiet
dc.contributor.authorLiu, Daniel S.
dc.contributor.authorWhite, Katharine Alice
dc.contributor.authorThompson, Samuel M.
dc.contributor.authorTing, Alice Y.
dc.date.accessioned2012-03-01T19:00:24Z
dc.date.available2012-03-01T19:00:24Z
dc.date.issued2009-10
dc.identifier.issn0002-7863
dc.identifier.issn1520-5126
dc.identifier.urihttp://hdl.handle.net/1721.1/69549
dc.description.abstractEscherichia coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine side chains of three acceptor proteins involved in oxidative metabolism. Our lab has shown that LplA and engineered mutants can ligate useful small-molecule probes such as alkyl azides ( Nat. Biotechnol. 2007, 25, 1483−1487) and photo-cross-linkers ( Angew. Chem., Int. Ed. 2008, 47, 7018−7021) in place of lipoic acid, facilitating imaging and proteomic studies. Both to further our understanding of lipoic acid metabolism, and to improve LplA’s utility as a biotechnological platform, we have engineered a novel 13-amino acid peptide substrate for LplA. LplA’s natural protein substrates have a conserved β-hairpin structure, a conformation that is difficult to recapitulate in a peptide, and thus we performed in vitro evolution to engineer the LplA peptide substrate, called “LplA Acceptor Peptide” (LAP). A 107 library of LAP variants was displayed on the surface of yeast cells, labeled by LplA with either lipoic acid or bromoalkanoic acid, and the most efficiently labeled LAP clones were isolated by fluorescence activated cell sorting. Four rounds of evolution followed by additional rational mutagenesis produced a “LAP2” sequence with a kcat/Km of 0.99 μM−1 min−1, >70-fold better than our previous rationally designed 22-amino acid LAP1 sequence (Nat. Biotechnol. 2007, 25, 1483−1487), and only 8-fold worse than the kcat/Km values of natural lipoate and biotin acceptor proteins. The kinetic improvement over LAP1 allowed us to rapidly label cell surface peptide-fused receptors with quantum dots.en_US
dc.language.isoen_US
dc.publisherAmerican Chemical Societyen_US
dc.relation.isversionofhttp://dx.doi.org/10.1021/ja904596fen_US
dc.rightsArticle is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.en_US
dc.sourceProf. Ting via Erja Kajosaloen_US
dc.titleYeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligaseen_US
dc.typeArticleen_US
dc.identifier.citationPuthenveetil, Sujiet et al. “Yeast Display Evolution of a Kinetically Efficient 13-Amino Acid Substrate for Lipoic Acid Ligase.” Journal of the American Chemical Society 131.45 (2009): 16430–16438.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemistryen_US
dc.contributor.approverTing, Alice Y.
dc.contributor.mitauthorTing, Alice Y.
dc.contributor.mitauthorPuthenveetil, Sujiet
dc.contributor.mitauthorLiu, Daniel S.
dc.contributor.mitauthorWhite, Katharine Alice
dc.contributor.mitauthorThompson, Samuel M.
dc.relation.journalJournal of the American Chemical Societyen_US
dc.eprint.versionAuthor's final manuscripten_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.orderedauthorsPuthenveetil, Sujiet; Liu, Daniel S.; White, Katharine A.; Thompson, Samuel; Ting, Alice Y.en
dc.identifier.orcidhttps://orcid.org/0000-0002-8277-5226
mit.licensePUBLISHER_POLICYen_US
mit.metadata.statusComplete


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