Document Detail


Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase.
MedLine Citation:
PMID:  19863063     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Escherichia 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 beta-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 approximately 10(7) 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 k(cat)/K(m) of 0.99 muM(-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 k(cat)/K(m) 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.
Authors:
Sujiet Puthenveetil; Daniel S Liu; Katharine A White; Samuel Thompson; Alice Y Ting
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of the American Chemical Society     Volume:  131     ISSN:  1520-5126     ISO Abbreviation:  J. Am. Chem. Soc.     Publication Date:  2009 Nov 
Date Detail:
Created Date:  2009-11-11     Completed Date:  2010-02-12     Revised Date:  2013-05-31    
Medline Journal Info:
Nlm Unique ID:  7503056     Medline TA:  J Am Chem Soc     Country:  United States    
Other Details:
Languages:  eng     Pagination:  16430-8     Citation Subset:  IM    
Affiliation:
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Amino Acids / chemistry,  metabolism*
Catalysis
Escherichia coli / enzymology
Kinetics
Ligases / chemistry,  metabolism*
Oxidation-Reduction
Peptides / chemistry,  metabolism*
Protein Engineering
Saccharomyces cerevisiae / chemistry,  cytology,  metabolism*
Surface Properties
Thioctic Acid / chemistry,  metabolism*
Grant Support
ID/Acronym/Agency:
PN2 EY018244/EY/NEI NIH HHS; R01 GM072670-05/GM/NIGMS NIH HHS; R01GM072670/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Amino Acids; 0/Peptides; 62-46-4/Thioctic Acid; EC 6.-/Ligases
Comments/Corrections

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