Yeast Display Evolution of a Kinetically Efficient 13-Amino Acid Substrate for Lipoic Acid Ligase

Puthenveetil, Sujiet; Liu, Daniel S.; White, Katharine A.; Thompson, Samuel; Ting, Alice Y.

doi:10.1021/ja904596f

Cited by 95 publications

(104 citation statements)

References 49 publications

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“…Instead of E2p, the HPLC assay was performed with an "LplA acceptor peptide" (LAP). Specifically, we used the yeast display-evolved sequence "LAP4.3D" (20) due to its superior chromatographic properties on HPLC when fused to the carrier protein HP1 (11). Fig.…”

Section: Resultsmentioning

confidence: 99%

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A fluorophore ligase for site-specific protein labeling inside living cells

Uttamapinant

White

Baruah

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structureguided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.fluorescence microscopy | biotechnology | enzyme engineering T echniques for posttranslational labeling of proteins in living cells address some of the shortcomings of green fluorescent protein (GFP) by expanding the repertoire of chemical probes available for protein visualization (1). However, most of these methods, such as HaloTag (2), SNAP/CLIP (3), and DHFR (4), still use large protein tags that sterically interfere with protein trafficking and function, as GFP is known to do (5, 6). FlAsH (7) is the only peptide-based posttranslational labeling method that currently works inside living cells. At 12 amino acids, the FlAsH tag is much smaller than GFP, but poor labeling specificity (7-9), cellular toxicity, and undesired palmitoylation (7) and oxidation (8) of the Cys 4 recognition motif limit its utility.Here we introduce a method for protein labeling that utilizes a peptide tag while preserving high sequence specificity inside living cells. Our method, called PRIME (PRobe Incorporation Mediated by Enzymes), is based on a "fluorophore ligase" that is engineered from the Escherichia coli enzyme lipoic acid ligase (LplA). LplA's natural function is to ligate lipoic acid onto three E. coli proteins involved in oxidative metabolism (10). We previously used LplA for labeling of cell-surface proteins by demonstrating that the wild-type enzyme can ligate an azidoalkanoic acid instead of lipoic acid (11) (Fig. 1A, Middle). Ligated azide could then be chemoselectively derivatized using cyclooctynefluorophore conjugates.In this work, we wished to extend LplA-mediated labeling to intracellular proteins but recognized the challenges associated with our two-step labeling scheme. First, labeling sensitivity is limited by the kinetics of strain-promoted ½3 þ 2 cycloaddition, which has a rate constant of 4.3 × 10 −3 M −1 sec −1 (12). Second, for intracellular applications, two washout steps would be needed: first to remove excess azide, and second to remove excess fluorophore. We s...

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Section: Resultsmentioning

confidence: 99%

“…We proceeded to characterize the two most active coumarin ligases identified in our screen: W37V LplA and W37I LplA. First, HPLC assays were performed, this time using our most kinetically efficient LAP called "LAP2" (20). Controls in Fig.…”

Section: Resultsmentioning

confidence: 99%

A fluorophore ligase for site-specific protein labeling inside living cells

Uttamapinant

White

Baruah

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

269

315

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“…Biotin Ligase and Lipoic Acid Ligase. The Ting group pioneered the approach of redirecting biotin ligase 312 and lipoic acid ligase 313,314 for protein labeling. The E. coli biotin ligase BirA catalyzes the formation of an amide bond between the carboxylic group of biotin and the ε-amino of a lysine situated in a 23-amino-acid recognition sequence ( Figure 16b), also known as acceptor sequence.…”

Section: Chemoenzymatic Labeling Based On Posttranslational Modificatmentioning

confidence: 99%

Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics

2016

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The superfamily of G protein-coupled receptors (GPCRs) mediates a wide range of physiological responses and serves as an important category of drug targets. Earlier biochemical and biophysical studies have shown that GPCRs exist temporally in an ensemble of interchanging conformations. Single-molecule techniques are ideally suited to understand the dynamic signaling and conformational complexity of G protein-coupled receptors (GPCRs). Here, we review the progress in single-molecule studies on GPCRs. We introduce the fundamental technical aspects of single-molecule fluorescence. We also survey the methodologies for labeling GPCRs with biophysical probes, particularly fluorescent dyes, and highlight the relevant chemical biology innovations that can be instrumental for studying GPCRs. Finally, we illustrate how the optical techniques and the labeling schemes have been combined to investigate GPCR signaling and dynamics at the single-molecule level.

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“…Compared to other thermostabilizing quantitating methods, this technology does not require production and purification of antibodies, and is easy to perform in a highthroughput manner. Puthenveetil et al (2009) applied yeast display to screen lipoic acid ligase acceptor peptides. The best evolution was from a *10 7 library of ligase acceptor peptides displayed on the yeast surface.…”

Section: Apply In Enzyme Evolutionmentioning

confidence: 99%

Combination of site-directed mutagenesis and yeast surface display enhances Rhizomucor miehei lipase esterification activity in organic solvent

et al. 2011

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To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.

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Yeast Display Evolution of a Kinetically Efficient 13-Amino Acid Substrate for Lipoic Acid Ligase

Cited by 95 publications

References 49 publications

A fluorophore ligase for site-specific protein labeling inside living cells

A fluorophore ligase for site-specific protein labeling inside living cells

Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics

Combination of site-directed mutagenesis and yeast surface display enhances Rhizomucor miehei lipase esterification activity in organic solvent

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