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Selective amine labeling of cell surface proteins guided by coiled‐coil assembly
Author(s) -
Yano Yoshiaki,
Furukawa Nami,
Ono Satoshi,
Takeda Yuki,
Matsuzaki Katsumi
Publication year - 2016
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.22715
Subject(s) - chemistry , covalent bond , membrane , coiled coil , glycophorin , sodium dodecyl sulfate , fluorescence , membrane protein , biophysics , micelle , amine gas treating , site directed spin labeling , cell membrane , biochemistry , organic chemistry , aqueous solution , physics , quantum mechanics , biology
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.