Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

Dockalova, Veronika; Sánchez‐Carnerero, Esther M.; Dunajova, Zuzana; Palao, Eduardo; Slanska, Michaela; Buryška, Tomáš; Damborský, Jiřı́; Klán, Petr; Prokop, Zbyněk

doi:10.1016/j.csbj.2020.03.029

Cited by 13 publications

(11 citation statements)

References 43 publications

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“…In prior work, photocage testing was performed using a simple “dummy” carboxylic acid leaving group, typically acetic acid. Thus far, carboxylic acids, amines, alcohols, thiols, halides, xanthanes, and phenols − have been released using BODIPY photocages. However, the evaluation of the photorelease of these functional groups was performed with the “first generation” BODIPY photocages, which are much less efficient for caging acetic acid than the most recently developed BODIPY photocages.…”

Section: Introductionmentioning

confidence: 99%

Efficiency of Functional Group Caging with Second-Generation Green- and Red-Light-Labile BODIPY Photoremovable Protecting Groups

et al. 2022

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BODIPY-based photocages release substrates by excitation with wavelengths in the visible to near-IR regions. The recent development of more efficient BODIPY photocages spurred us to evaluate the scope and efficiency of these second-generation boron-methylated green-light and red-light-absorbing BODIPY photocages. Here, we show that these more photosensitive photocages release amine, alcohol, phenol, phosphate, halides, and carboxylic acid derivatives with much higher quantum yields than first-generation BODIPY photocages and excellent chemical yields. Chemical yields are near-quantitative for the release of all functional groups except the photorelease of amines, which react with concomitantly photogenerated singlet oxygen. In these cases, high chemical yields for photoreleased amines are restored by irradiation under an inert atmosphere. The photorelease quantum yield has a weak relationship with the leaving group pK a of the green-absorbing BODIPY photocages but little relationship with the red-absorbing derivatives, suggesting that factors other than leaving group quality impact the quantum yield. For the photorelease of alcohols, in all cases a carbonate linker (that loses CO2 upon photorelease) significantly increases both the quantum yield and the chemical yield compared to those for direct photorelease via the ether.

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Section: Introductionmentioning

confidence: 99%

Efficiency of Functional Group Caging with Second-Generation Green- and Red-Light-Labile BODIPY Photoremovable Protecting Groups

et al. 2022

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“…30 Subsequent mutagenesis in the p3 region (F143L, W149A, and I211L) opened a new tunnel and introduced additional internal flexibility of the cap domain (variant LinB86), resulting in the most proficient haloalkane dehalogenase reported to date for the reaction with 1,2-dibromoethane (k cat = 57 s -1 at 37 C and pH 8.6). 31 A fluorogenic substrate 4-(bromomethyl)-6,7-dimethoxycoumarin (Br-COU) 32 was employed as a ''bulky'' molecular probe to assess the effects of the tunnel engineering (Figures S8 and S9).…”

Section: Transient Kinetics and Thermodynamics Of Haloalkane Dehalogenasementioning

confidence: 99%

Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling

Hess

Dockalova

Kokkonen

et al. 2021

Chem

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“…A wide range of diverse HaloTag ligands have been designed and synthesized, offering a variety of properties (Figure B), e.g., improved photostability and brightness, high biocompatibility or fluorogenicity allowing “no-wash” labeling protocols − or providing specific affinity handles . Despite the great diversity of ligands used, most of their applications always utilize the same tag protein DhaAHT, without considering the choice of another protein partner for better recognition of a specific ligand.…”

Section: Introductionmentioning

confidence: 99%

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

Marques

Slanska

Chmelova

et al. 2022

JACS Au

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HaloTag labeling technology has introduced unrivaled potential in protein chemistry and molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational analysis of different reporters, a tetramethylrhodamine- and three 4-stilbazolium-based fluorescent ligands, we showed that the mechanism of incorporating different ligands depends both on the binding step and the efficiency of the chemical reaction. By studying the different haloalkane dehalogenases DhaA, LinB, and DmmA, we found that the architecture of the access tunnels is critical for the kinetics of both steps and the ligand specificity. We showed that highly efficient labeling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from the wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates the need for expensive and laborious protein engineering.

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Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

Cited by 13 publications

References 43 publications

Efficiency of Functional Group Caging with Second-Generation Green- and Red-Light-Labile BODIPY Photoremovable Protecting Groups

Efficiency of Functional Group Caging with Second-Generation Green- and Red-Light-Labile BODIPY Photoremovable Protecting Groups

Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

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