Use of an Asparaginyl Endopeptidase for Chemo-enzymatic Peptide and Protein Labeling

Transpeptidases are ideal biocatalysts for site-specific peptide and protein labeling, whereas reactions that target N-terminus cysteine with commercially available reagents have become common practice. However, a versatile approach that allows bioconjugation at the terminus of choice (N or C), while avoiding the use of backbone-modified substrates (e.g. depsipeptide) or large excess of reagent, is highly desirable. Aiming to meet these benchmarks, we have combined the advantages of asparaginyl endopeptidase (AEP) catalysis with a N-terminal cysteine trapping reaction and created a chemo-enzymatic labeling system. In this approach, polypeptide with a Asn-Cys-Leu recognition sequence are ligated with a counterpart possessing an N-terminal Gly-Leu by AEP; the byproduct Cys-Leu is subsequently trapped by a stable and inexpensive scavenger, 2-formyl phenylboronic acid (FPBA), to yield an inert thiazolidine derivative, thereby driving the reaction forward to product formation. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP ligation/FPBA coupling system was further demonstrated by site-specific labeling the N- or C-termini of various proteins.

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Site-Specific C-Terminal Labeling of Peptides and Proteins using Asparaginyl Endopeptidase in a Chemo-Enzymatic Sequence

10.26434/chemrxiv.9633032.v1 ◽

2019 ◽

Author(s):

Simon Tang ◽

Davide Cardella ◽

Alexander J. Lander ◽

Xuefei Li ◽

Yu-Hsuan Tsai ◽

...

Keyword(s):

Phenylboronic Acid ◽

Catalytic Efficiency ◽

Model Reaction ◽

Protein Labeling ◽

Recognition Sequence ◽

Site Specific ◽

Asparaginyl Endopeptidase ◽

Leaving Group ◽

Reaction Proceeds ◽

Potential Use

Asparaginyl endopeptides (AEP) are recognized for their catalytic efficiency, presenting as ideal tools for protein bioconjugation. However, the peptide ligation catalyzed by AEP is reversible. In an attempt to obtain high reaction yields, thiodepsipeptides have been used as substrates but found to be highly unstable, and labeling is only limited to the N-terminus. To maximize the potential use of AEP, here we developed a novel chemo-enzymatic sequence for protein bioconjugation at both the N- and C-termini. In this system, an alternative recognition sequence, Asn-Cys-Leu, was used. Upon ligation, the reaction yields Cys-Leu as leaving group, and its reactive 1,2-aminothiol functionality was quenched by an effective and affordable electrophile, 2-formyl phenylboronic acid (FPBA), to yield a non-reactive cyclic byproduct. In the presence of FPBA our model reaction proceeds with ~95% yield using only 1.2 equivalent of substrate, whereas the yield remains at ~50% in the absence of this additive. This “quenching” approach enables protein labeling at both the N- and C-termini ranging from 75 to 85% (five examples). The simplicity and versatility of this quenching approach will enhance the future use of AEPs in protein bioconjugation.

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Lysine Acylation Using Conjugating Enzymes (LACE) for Site-Specific Modification and Ubiquitination of Native Proteins

10.26434/chemrxiv.10028900 ◽

2019 ◽

Author(s):

Raphael Hofmann ◽

Gaku Akimoto ◽

Thomas G. Wucherpfennig ◽

Cathleen Zeymer ◽

Jeffrey Bode

Keyword(s):

Protein Labeling ◽

Sequence Specificity ◽

Reaction Conditions ◽

One Pot ◽

Site Specific ◽

Protein Substrates ◽

Ubiquitinated Proteins ◽

Native Proteins ◽

Lysine Residues ◽

Substrate Tolerance

Enzymes are powerful tools for post-translational protein labeling due to their high sequence specificity and mild reaction conditions. Many existing protocols, however, are restricted to conjugations at terminal positions or rely on non-peptidic metabolites and large recognition domains. Here we introduce a chemoenzymatic method to functionalize proteins at internal lysine residues that are part of genetically encoded minimal recognition tags (four residues). We achieved this by employing the intrinsic sequence specificity of the E2 SUMO-conjugating enzyme Ubc9 and a short peptide thioester, which together obviate the need for E1 and E3 enzymes. Using a range of protein substrates, we apply this approach to the conjugation of biochemical probes, one-pot dual-labeling reactions in combination with sortase, and site-specific monoubiquitination and ISG15ylation. The small tag size and large substrate tolerance of Ubc9 will make this a method of choice for protein engineering by isopeptide formation and the preparation of ubiquitinated proteins.

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Lysine Acylation Using Conjugating Enzymes (LACE) for Site-Specific Modification and Ubiquitination of Native Proteins

10.26434/chemrxiv.10028900.v1 ◽

2019 ◽

Author(s):

Raphael Hofmann ◽

Gaku Akimoto ◽

Thomas G. Wucherpfennig ◽

Cathleen Zeymer ◽

Jeffrey Bode

Keyword(s):

Protein Labeling ◽

Sequence Specificity ◽

Reaction Conditions ◽

One Pot ◽

Site Specific ◽

Protein Substrates ◽

Ubiquitinated Proteins ◽

Native Proteins ◽

Lysine Residues ◽

Substrate Tolerance

Enzymes are powerful tools for post-translational protein labeling due to their high sequence specificity and mild reaction conditions. Many existing protocols, however, are restricted to conjugations at terminal positions or rely on non-peptidic metabolites and large recognition domains. Here we introduce a chemoenzymatic method to functionalize proteins at internal lysine residues that are part of genetically encoded minimal recognition tags (four residues). We achieved this by employing the intrinsic sequence specificity of the E2 SUMO-conjugating enzyme Ubc9 and a short peptide thioester, which together obviate the need for E1 and E3 enzymes. Using a range of protein substrates, we apply this approach to the conjugation of biochemical probes, one-pot dual-labeling reactions in combination with sortase, and site-specific monoubiquitination and ISG15ylation. The small tag size and large substrate tolerance of Ubc9 will make this a method of choice for protein engineering by isopeptide formation and the preparation of ubiquitinated proteins.

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A Site-Specific Bifunctional Protein Labeling System for Affinity and Fluorescent Analysis

Bioconjugate Chemistry ◽

10.1021/bc050213j ◽

2005 ◽

Vol 16 (6) ◽

pp. 1352-1355 ◽

Cited By ~ 5

Author(s):

Thomas S. Shute ◽

Masayuki Matsushita ◽

Tobin J. Dickerson ◽

James J. La Clair ◽

Kim D. Janda ◽

...

Keyword(s):

Protein Labeling ◽

Site Specific ◽

Bifunctional Protein ◽

Fluorescent Analysis ◽

A Site ◽

Labeling System

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Enzyme-based protein-tagging systems for site-specific labeling of proteins in living cells

Microscopy ◽

10.1093/jmicro/dfaa011 ◽

2020 ◽

Vol 69 (3) ◽

pp. 156-166 ◽

Cited By ~ 1

Author(s):

Shinji Sueda

Keyword(s):

Fluorescent Protein ◽

Protein Labeling ◽

Specific Interactions ◽

Post Translational Modification ◽

Site Specific ◽

Target Proteins ◽

Enzyme Reactions ◽

Substrate Protein ◽

Labeling System ◽

Protein Tagging

Abstract Various protein-labeling methods based on the specific interactions between genetically encoded tags and synthetic probes have been proposed to complement fluorescent protein-based labeling. In particular, labeling methods based on enzyme reactions have been intensively developed by taking advantage of the highly specific interactions between enzymes and their substrates. In this approach, the peptides or proteins are genetically attached to the target proteins as a tag, and the various labels are then incorporated into the tags by enzyme reactions with the substrates carrying those labels. On the other hand, we have been developing an enzyme-based protein-labeling system distinct from the existing ones. In our system, the substrate protein is attached to the target proteins as a tag, and the labels are incorporated into the tag by post-translational modification with an enzyme carrying those labels followed by tight complexation between the enzyme and the substrate protein. In this review, I summarize the enzyme-based protein-labeling systems with a focus on several typical methods and then describe our labeling system based on tight complexation between the enzyme and the substrate protein.

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Suzuki–Miyaura cross-coupling for chemoproteomic applications

10.26434/chemrxiv.12055218.v1 ◽

2020 ◽

Author(s):

Jian Cao ◽

Ernest Armenta ◽

Lisa Boatner ◽

Heta Desai ◽

Neil Chan ◽

...

Keyword(s):

Mass Spectrometry ◽

Cross Coupling ◽

Protein Profiling ◽

Protein Labeling ◽

Caspase 8 ◽

Complex Cell ◽

Bioorthogonal Chemistry ◽

Reaction Conditions ◽

Target Deconvolution ◽

Palladium Catalyzed

Bioorthogonal chemistry is a mainstay of chemoproteomic sample preparation workflows. While numerous transformations are now available, chemoproteomic studies still rely overwhelmingly on copper-catalyzed azide –alkyne cycloaddition (CuAAC) or 'click' chemistry. Here we demonstrate that gel-based activity-based protein profiling (ABPP) and mass-spectrometry-based chemoproteomic profiling can be conducted using Suzuki–Miyaura cross-coupling. We identify reaction conditions that proceed in complex cell lysates and find that Suzuki –Miyaura cross-coupling and CuAAC yield comparable chemoproteomic coverage. Importantly, Suzuki–Miyaura is also compatible with chemoproteomic target deconvolution, as demonstrated using structurally matched probes tailored to react with the cysteine protease caspase-8. Uniquely enabled by the observed orthogonality of palladium-catalyzed cross-coupling and CuAAC, we combine both reactions to achieve dual protein labeling.

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