Site‐Specific Labelling of Native Mammalian Proteins for Single‐Molecule FRET Measurements

Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states, and modifications, such as phosphorylation, affect their function. Hence, u...

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Bibliographic Details
Published in:Chembiochem : a European journal of chemical biology 2018-04, Vol.19 (8), p.780-783
Main Authors: Gust, Alexander, Jakob, Leonhard, Zeitler, Daniela M., Bruckmann, Astrid, Kramm, Kevin, Willkomm, Sarah, Tinnefeld, Philip, Meister, Gunter, Grohmann, Dina
Format: Article
Language:English
Subjects:
Argonaute
Argonaute 2 protein
Biomolecules
bioorganic chemistry
Cellular structure
Coupling (molecular)
Dyes
Fluorescence
Fluorescence resonance energy transfer
Fluorescent dyes
Fluorescent indicators
FRET
Labeling
Mammals
Phosphorylation
Proteins
Proteomes
single-molecule studies
Staudinger ligation
Structure-function relationships
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Summary:Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states, and modifications, such as phosphorylation, affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single‐molecule FRET measurements allow exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome, as a method for the site‐specific coupling of organic dyes into native, non‐recombinant mammalian proteins is lacking. We address this issue showing the site‐specific engineering of fluorescent dyes into human proteins on the basis of bioorthogonal reactions. We show the applicability of the method to study functional and post‐translationally modified proteins on the single‐molecule level, among them the hitherto inaccessible human Argonaute 2. Don't FRET! We describe a method that enables fast site‐specific fluorescent labelling of human proteins produced in their endogenous cellular environment with external chemical groups such as fluorescent dyes and an experimental strategy to make the labelled proteins amenable for single‐molecule FRET analysis under near‐physiological conditions.
ISSN:1439-4227
1439-7633
DOI:10.1002/cbic.201700696