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Site‐Specific Labelling of Native Mammalian Proteins for Single‐Molecule FRET Measurements
Author(s) -
Gust Alexander,
Jakob Leonhard,
Zeitler Daniela M.,
Bruckmann Astrid,
Kramm Kevin,
Willkomm Sarah,
Tinnefeld Philip,
Meister Gunter,
Grohmann Dina
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700696
Subject(s) - bioorthogonal chemistry , förster resonance energy transfer , biomolecule , human proteome project , single molecule fret , chemistry , proteome , function (biology) , biophysics , fluorescence , computational biology , biochemistry , proteomics , biology , microbiology and biotechnology , combinatorial chemistry , click chemistry , physics , quantum mechanics , gene
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.