Details

Autor(en) / Beteiligte

• Gust, Alexander
• Jakob, Leonhard
• Zeitler, Daniela M.
• Bruckmann, Astrid
• Kramm, Kevin
• Willkomm, Sarah
• Tinnefeld, Philip
• Meister, Gunter
• Grohmann, Dina

Titel

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

Ist Teil von

• Chembiochem : a European journal of chemical biology, 2018-04, Vol.19 (8), p.780-783

Ort / Verlag

Germany: Wiley Subscription Services, Inc

Erscheinungsjahr

2018

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.