Enlightening the Path to Protein Engineering: Chemoselective Turn-On Probes for High-Throughput Screening of Enzymatic Activity

Abstract

Many successful stories in enzyme engineering are based on the creation of randomized diversity in large mutant libraries, containing millions to billions of enzyme variants. Methods that enabled their evaluation with high throughput are dominated by spectroscopic techniques due to their high speed and sensitivity. A large proportion of studies relies on fluorogenic substrates that mimic the chemical properties of the target or coupled enzymatic assays with an optical read-out that assesses the desired catalytic efficiency indirectly. The most reliable hits, however, are achieved by screening for conversions of the starting material to the desired product. For this purpose, functional group assays offer a general approach to achieve a fast, optical read-out. They use the chemoselectivity, differences in electronic and steric properties of various functional groups, to reduce the number of false-positive results and the analytical noise stemming from enzymatic background activities. This review summarizes the developments and use of functional group probes for chemoselective derivatizations, with a clear focus on screening for enzymatic activity in protein engineering.

Ein universeller, kontinuierlicher Assay für SAM‐abhängige Methyltransferasen

“…Beim Screening auf neuartige Aktivitäten (die in den ersten Screening-Runden voraussichtlich gering sein werden) sind sowohl die Empfindlichkeit als auch die Fähigkeit, in Zelllysaten zu screenen, von entscheidender Bedeutung. [24] Ein von Tan et al beschriebener Homocystein-Assay ermöglicht den selektiven Nachweis von L-Homocystein gegenüber Cystein im Plasma über die Bildung von Schwefelwasserstoff durch eine Homocystein-α,γ-Lyase. [25] Der Fluoreszenznachweis mit N,N-Dibutylphenylendiamin…”

“…Aus diesem Grund führen andere biologische Thiole wie Glutathion und Cystein oder exogen zugesetzte Reduktionsmittel wie 2‐Mercaptoethanol zu einem hohen Hintergrund, was die Empfindlichkeit des Assays verringert und für die Verwendung mit Zelllysaten inkompatibel macht. Beim Screening auf neuartige Aktivitäten (die in den ersten Screening‐Runden voraussichtlich gering sein werden) sind sowohl die Empfindlichkeit als auch die Fähigkeit, in Zelllysaten zu screenen, von entscheidender Bedeutung [24] …”

A Universal, Continuous Assay for SAM‐dependent Methyltransferases

“…When screening for novel activities (which are expected to be low during initial rounds of screening), sensitivity and the ability to screen in cell lysates are both critically important. [24] One homocysteine assay reported by Tan et al features selective detection of L-homocysteine over cysteine in plasma through formation of hydrogen sulfide by a homocysteine-α,γ-lyase. [25] Fluorescent detection with N,N-dibutyl phenylene diamine (DBPDA) serves as the sensitive readout (Scheme 1E).…”

Naphthyridine-Based Electron Push–Pull-Type Amine-Reactive Fluorescent Probe for Sensing Amines and Proteins in Aqueous Media

“…In fluorescence bioimaging, the amine group is an extremely attractive target functional group because of its ubiquity and high reactivity. Many “turn-on type” amine-reactive fluorescent probes bearing active esters, isothiocyanates, and sulfonyl chlorides as amine-reactive groups have been reported for valid fluorescence labeling . The requirements of labeled reagents are fast and reliable chemical reactions and a compact molecular size for the exclusion of perturbations of the targeting biomolecules.…”

“…Many "turn-on type" aminereactive fluorescent probes bearing active esters, isothiocyanates, and sulfonyl chlorides as amine-reactive groups have been reported for valid fluorescence labeling. 2 The requirements of labeled reagents are fast and reliable chemical reactions and a compact molecular size for the exclusion of perturbations of the targeting biomolecules. Bicyclic fluorophores, such as coumarin and 7-nitrobenzoxadiazole (NBD), are widely utilized as typical amine-reactive fluorophores with compact sizes.…”