Orientation of the pore-forming peptide GALA in POPC vesicles determined by a BODIPY-avidin/biotin binding assay.
Orientation of the pore-forming peptide GALA in POPC vesicles determined by a BODIPY-avidin/biotin binding assay.
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DOI：
10.1016/s0006-3495(99)77368-6
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发表时间：
1999-04
影响因子：
3.4
通讯作者：
F. Nicol;S. Nir;F. Szoka
中科院分区：
文献类型：
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作者：
F. Nicol;S. Nir;F. Szoka
我们利用BODIPY-Avidin与生物素部分不可逆结合后的荧光增强，在pH 5.0的大单层磷脂酰胆碱囊泡中确定了生物素化版本的造孔肽Gala(WEALAEALAEALAEHLAEALAEALAEALEALAA)的取向。Gala及其变异体在N-末端或C-末端被生物素化。BODIPY-Avidin被添加到外部或预包埋在囊泡中，以评估分别将其标记末端暴露于双层外侧或内侧的脂质体结合的生物素化Gala的比例。在大多数膜结合肽以跨膜聚集体形式存在并形成水孔的条件下(脂质/结合肽的摩尔比为2500/1)，膜结合肽的头尾(N-端到C-端)方向是这样的：多肽的N-端暴露在囊泡的内侧，C-端暴露在囊泡的外部。在导致气孔形成减少的条件下(较高的脂/肽摩尔比)，我们观察到暴露在囊泡外部的GalA末端的比例增加。这些结果与一个模型(Parente等人，生物化学，29：8720,1990)一致，该模型要求聚集体中的临界数量的多肽(M)形成跨双层结构。当多肽形成Szei聚集体时，当M=4~6时，多肽的取向大部分平行于膜表面，使得生物素化多肽的两端都暴露在外部的BODIPY-Avidin中。这种BODIPY-亲和素/生物素结合分析应该有助于确定其他膜相互作用分子的取向。
We determined the orientation of a biotinylated version of the pore-forming peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) at pH 5.0 in large unilamellar phosphatidylcholine vesicles, using the enhancement of BODIPY-avidin fluorescence subsequent to its irreversible binding to a biotin moiety. GALA and its variants were biotinylated at the N- or C-terminus. BODIPY-avidin was either added externally or was pre-encapsulated in vesicles to assess the fraction of liposome-bound biotinylated GALA that exposed its labeled terminus to the external or internal side of the bilayer, respectively. Under conditions where most of the membrane-bound peptides were involved in transmembrane aggregates and formed aqueous pores (at a lipid/bound peptide molar ratio of 2500/1), the head-to-tail (N- to C-terminus) orientation of the membrane-inserted peptides was such thatof the peptides exposed their N-terminus on the inside of the vesicle and their C-terminus on the outside. Under conditions resulting in reduced pore formation (at higher lipid/peptide molar ratios), we observed an increase in the fraction of GALA termini exposed to the outside of the vesicle. These results are consistent with a model (Parente et al., Biochemistry, 29:8720, 1990) that requires a critical number of peptides (M) in an aggregate to form a transbilayer structure. When the peptides form an aggregate of sizei, withi<M=4 to 6, the orientation of the peptides is mostly parallel to the membrane surface, such that both termini of the biotinylated peptide are exposed to external BODIPY-avidin. This BODIPY-avidin/biotin binding assay should be useful to determine the orientation of other membrane-interacting molecules.