Selective amine labeling of cell surface proteins guided by coiled-coil assembly.
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.
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-Ku, Kyoto, 606-8501, Japan.
Yoshiaki
Y
Yano
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-Ku, Kyoto, 606-8501, Japan.
Nami
N
Furukawa
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-Ku, Kyoto, 606-8501, Japan.
Satoshi
S
Ono
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-Ku, Kyoto, 606-8501, Japan.
Yuki
Y
Takeda
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-Ku, Kyoto, 606-8501, Japan.
Katsumi
K
Matsuzaki
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D006021:Glycophorins / Q000096:biosynthesis / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*
D006021:Glycophorins; D012967:Sodium Dodecyl Sulfate; D002784:Cholesterol
coiled-coil; fluorescent probes; membrane proteins