Kinetic and structural characterization of the self-labeling protein tags HaloTag7, SNAP-tag, and CLIP-tag
| Autor: | Jochen Reinstein, Jana Tünnermann, Guillaume Gotthard, Nicole Mertes, Stefanie Kühn, Ulrike Uhrig, Lin Xue, Kai Johnsson, Timo Tänzer, Jonas Wilhelm, Julien Hiblot, Miroslaw Tarnawski, Julie Karpenko |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Models
Molecular general-method Recombinant Fusion Proteins Protein tag fluorescent sensor Biochemistry Article Substrate Specificity Rhodamine O(6)-Methylguanine-DNA Methyltransferase chemistry.chemical_compound superresolution microscopy fluorophores haloalkane dehalogenases directed evolution Fluorescent Dyes Staining and Labeling Rhodamines Substrate (chemistry) Orders of magnitude (angular velocity) Directed evolution Combinatorial chemistry Fluorescence o-6-alkylguanine-dna alkyltransferase Protein Structure Tertiary SNAP-tag Kinetics fluorogenic probes chemistry Covalent bond live-cell fusion proteins |
| Zdroj: | Biochemistry Biochemistry, 60 (33) 'Biochemistry ', vol: 60, pages: 2560-2575 (2021) |
| ISSN: | 0006-2960 1520-4995 |
| Popis: | The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics. Biochemistry, 60 (33) ISSN:0006-2960 ISSN:1520-4995 |
| Databáze: | OpenAIRE |
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