Selective amine labeling of cell surface proteins guided by coiled-coil assembly
| Autor: | Yoshiaki Yano, Satoshi Ono, Yuki Takeda, Nami Furukawa, Katsumi Matsuzaki |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Biophysics 010402 general chemistry 01 natural sciences Biochemistry Biomaterials Cell membrane 03 medical and health sciences chemistry.chemical_compound medicine Glycophorin Sodium dodecyl sulfate Coiled coil biology Organic Chemistry Peripheral membrane protein General Medicine 0104 chemical sciences 030104 developmental biology Membrane medicine.anatomical_structure chemistry Membrane protein Covalent bond biology.protein |
| Zdroj: | Biopolymers. 106:484-490 |
| ISSN: | 0006-3525 |
| DOI: | 10.1002/bip.22715 |
| Popis: | Covalent labeling of target proteins in living cells is useful for both fluorescence live-cell imaging and the subsequent biochemical analyses of the proteins. Here, we report an efficient method for the amine labeling of membrane proteins on the cell surface, guided by a noncovalent coiled-coil interaction. A carboxyl sulfosuccinimidyl ester introduced at the C-terminus of the coiled-coil probe reacted with target proteins under mild labeling conditions ([probe] = 150 nM, pH 7.4, 25°C) for 20 min. Various fluorescent moieties with different hydrophobicities are available for covalent labeling with high signal/background labeling ratios. Using this method, oligomeric states of glycophorin A (GpA) were compared in mammalian CHO-K1 cells and sodium dodecyl sulfate (SDS) micelles. In the cell membranes, no significant self-association of GpA was detected, whereas SDS-PAGE suggested partial dimerization of the proteins. Membrane cholesterol was found to be an important factor that suppressed the dimerization of GpA. Thus, the covalent functionality enables direct comparison of the oligomeric state of membrane proteins under various conditions. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 484-490, 2016. |
| Databáze: | OpenAIRE |
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