To normalize for variations in expression level, the yeast pool is also collectively labeled with anti-c-Myc antibody, detected with a secondary antibody conjugated to Alexa Fluor 488, which is very easily resolvable from phycoerythrin fluorescence. 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 development followed by additional rational mutagenesis produced a LAP2 sequence with a kcat/Kmof 0.99 M1min1, >70-fold better than our previous rationally-designed 22-amino acid LAP1 sequence (Nat. Biotechnol.2007,25, 14831487), and only 8-fold worse than the kcat/Kmvalues 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. == Introduction == Most proteins are developed to interact with a multitude of cellular molecules and thus contain a quantity of unique domains, binding sites, and activities. Often, it is useful to the biochemist to reduce a specific aspect of a proteins function to just a peptide fragment. This can help to determine the minimal features of a protein required for a specific function such as binding, acknowledgement by an enzyme, translocation, or folding.14It may also be desirable to create a consensus peptide substrate for p53 and MDM2 proteins-interaction-inhibitor racemic assay purposes,5;6or to use a peptide in place p53 and MDM2 proteins-interaction-inhibitor racemic of a protein to facilitate crystallography of multi-protein complexes.7;8For therapeutic applications, replacement of protein drugs with peptides having comparable activity can improve tissue penetration and reduce immunogenicity.9;10Our lab is interested in protein minimization to peptides for the purpose of developing new protein labeling technologies. Size minimization of protein tags that direct the targeting of fluorescent probes11can greatly reduce problems of tag interference with protein trafficking, folding, and interactions. Conversion of proteins to peptides without loss of the function of interest, however, is usually challenging for a number of reasons. First, the function may require secondary structure that is hard to recapitulate in a peptide. Second, the function may require contributions from multiple, noncontiguous regions of a protein. Third, structural information is not available for many Rabbit Polyclonal to PTPN22 proteins, and in some cases, even the regions that contribute to a proteins relevant activity are not known. Fourth, due to their more flexible structure, peptide binding is usually often associated with a greater entropic penalty than is usually protein binding12, making it more difficult to engineer high-affinity interactions. Numerous methods have been used to reduce proteins to peptides. Simple truncation and/or rational design can be successful,1315but is usually associated with at least a partial loss of activity and/or specificity. Peptide scanning16or high-throughput screening1719approaches are more exhaustive, p53 and MDM2 proteins-interaction-inhibitor racemic but library sizes are limited (typically 102 105), so it is difficult to identify optimal sequences. Peptideselections, on the other hand, can process libraries up to 109in size, dramatically increasing the probability of identifying a successful sequence. Accordingly, selections on phage,2023inside bacteria,24and on the surface of bacteria,25yeast,26and mammalian cells,27have been used to evolve peptides with novel functions. In this study, our goal was to identify novel, kinetically efficient peptide substrates forE. colilipoic acid ligase (LplA) (Physique 1). LplA is usually a cofactor ligase that our lab has harnessed for fluorescent protein labeling applications.13;28The natural function of LplA is to catalyze ATP-dependent, covalent ligation of lipoic acid (Figure 1A) onto specific lysine sidechains of threeE. coliproteins involved in oxidative metabolism: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and the glycine cleavage system.29Previously, we showed that LplA and engineered variants could ligate unnatural probes such as an alkyl azide (a functional group handle for fluorophore introduction;Physique 1A),13a fluorinated aryl azide photocrosslinker,28bromoalkanoic acid (a ligand for HaloTag30;Physique 1A),31and a coumarin fluorophore32in place of lipoic acid. To utilize these ligation reactions for protein imaging applications, we prepared recombinant fusions of proteins of interest (POIs) to the 9 kD E2p domain name of pyruvate dehydrogenase (Physique 1Btop).13Such fusions.