Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center , 1275 York Avenue, New York, New York 10065, United States.
Program of Pharmacology, Weill Graduate School of Medical Sciences of Cornell University , New York, New York 10021, United States.
Langmuir. 2017 Mar 28;33(12):3028-3039. doi: 10.1021/acs.langmuir.6b04150. Epub 2017 Mar 14.
The popularization of studies in membrane protein lipid phase coexistence has prompted the development of new techniques to construct and study biomimetic systems with cholesterol-rich lipid microdomains. Here, microsphere-supported biomembranes with integrated α-helical peptides, referred to as proteolipobeads (PLBs), were used to model peptide/protein partitioning within DOPC/DPPC/cholesterol phase-separated membranes. Due to the appearance of compositional heterogeneity and impurities in the formation of model PLB assemblies, fluorescence-activated cell sorting (FACS) was used to characterize and sort PLB populations on the basis of disordered phase (L) content. In addition, spectral imaging was used to assess the partitioning of FITC-labeled α-helical peptide between fluorescently labeled L phase and unlabeled ordered phase (L) phase lipid microdomains. The apparent peptide partition coefficient, K, was measured to be 0.89 ± 0.06, indicating a slight preference of the peptide for the L phase. A biomimetic motif of the L phase concentration enhancement of the biotinyl-peptide ligand display in proteolipobeads was also observed. Finally, peptide mobility was measured by FRAP separately in each lipid phase, yielding diffusivities of 0.036 ± 0.005 and 0.014 ± 0.003 μm/s in the L and L phases, respectively.
膜蛋白脂相共存研究的普及促使人们开发新的技术来构建和研究富含胆固醇的脂微区的仿生系统。在这里，使用带有整合α-螺旋肽的微球支撑的生物膜，称为脂肽小球（PLB），来模拟 DOPC/DPPC/胆固醇相分离膜中肽/蛋白的分区。由于模型 PLB 组装形成过程中存在组成异质性和杂质，因此使用荧光激活细胞分选（FACS）基于无序相（L）含量对 PLB 群体进行表征和分选。此外，光谱成像用于评估 FITC 标记的α-螺旋肽在荧光标记的 L 相和未标记的有序相（L）相脂质微区之间的分区。测得的明显肽分配系数 K 为 0.89±0.06，表明肽对 L 相略有偏好。还观察到生物素化肽配体在脂肽小球中显示的 L 相浓度增强的仿生基序。最后，通过 FRAP 分别在每个脂质相中测量肽的流动性，在 L 和 L 相中分别得到 0.036±0.005 和 0.014±0.003 μm/s 的扩散系数。
