Frey S, Tamm L K
Department of Biophysical Chemistry, Biocenter, University of Basel, Switzerland.
Biochem J. 1990 Dec 15;272(3):713-9. doi: 10.1042/bj2720713.
The synthetic 25-residue signal peptide of cytochrome c oxidase subunit IV was labelled with the fluorophor 7-nitrobenz-2-oxa-1,3-diazole (NBD) at its single cysteine residue. Addition of small unilamellar vesicles of 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) to the labelled peptide resulted in a shift of the NBD excitation and emission spectra to shorter wavelengths. Binding of the peptide to the vesicles was measured by the increase in the fluorescence emission yield. A surface partition constant of (3.9 +/- 0.5) x 10(3) M-1 was derived from these titrations. When the membrane contained, in addition to POPC, negatively charged 1-palmitoyl 2-oleoyl phosphatidylglycerol (POPG), the NBD fluorescence spectra were further shifted to shorter wavelengths and exhibited increased quantum yields. The apparent partition constants were increased to 10(4)-10(5) M-1 for vesicles with 20 or 100 mol% POPG. Lateral diffusion of the peptide was measured by fluorescence recovery after photobleaching in multibilayers of POPC, POPG, POPC/POPG (4:1) and 1,2-dimyristoyl phosphatidylcholine. The lateral diffusion coefficients of the peptide in bilayers of POPC (8 x 10(-8) cm2/s at 21 degrees C) were 1.5-1.6-fold greater than those of NBD-labelled phospholipids (5 x 10(-8) cm2/s at 21 degrees C), but 1.5-1.8-fold smaller (3 x 10(-8) cm2/s in 20% POPG and at 21 degrees C) than the lipid diffusion coefficients in the negatively charged bilayers. It is concluded that the signal peptide associates with phospholipid bilayers in two different forms, which depend on the lipid charge. The experiments with POPC bilayers are well explained by a model in which the peptide partitions into the region of the phospholipid head-groups and diffuses along the membrane/water interface. If POPG is present in the membrane, electrostatic attractions between the basic residues of the peptide and the acidic lipid head-groups result in a deeper penetration of the bilayer. For this case, two models that are both consistent with the experimental data are discussed, in which the peptide either forms an oligomer of three to six partially helical membrane-spanning monomers, or inserts into the bilayer with its amphiphilic helical segment aligned parallel to the plane of the membrane and located near the head-group and outer hydrocarbon region of the bilayer.
细胞色素c氧化酶亚基IV的25个氨基酸残基的合成信号肽在其单个半胱氨酸残基处用荧光团7-硝基苯并-2-恶唑-1,3-二氮杂茂（NBD）进行标记。向标记的肽中添加1-棕榈酰-2-油酰磷脂酰胆碱（POPC）的小单层囊泡，导致NBD激发和发射光谱向较短波长移动。通过荧光发射产率的增加来测量肽与囊泡的结合。从这些滴定中得出表面分配常数为(3.9±0.5)×10³ M⁻¹。当膜中除了POPC外还含有带负电荷的1-棕榈酰-2-油酰磷脂酰甘油（POPG）时，NBD荧光光谱进一步向较短波长移动并表现出量子产率增加。对于含有20或100 mol% POPG的囊泡，表观分配常数增加到10⁴ - 10⁵ M⁻¹。通过在POPC、POPG、POPC/POPG（4:1）和1,2-二肉豆蔻酰磷脂酰胆碱的多层膜中进行光漂白后的荧光恢复来测量肽的横向扩散。肽在POPC双层膜中的横向扩散系数（21℃时为8×10⁻⁸ cm²/s）比NBD标记的磷脂（21℃时为5×10⁻⁸ cm²/s）大1.5 - 1.6倍，但比带负电荷双层膜中的脂质扩散系数小1.5 - 1.8倍（20% POPG且21℃时为3×10⁻⁸ cm²/s）。得出的结论是，信号肽以两种不同形式与磷脂双层结合，这取决于脂质电荷。用POPC双层膜进行的实验可以很好地用一个模型来解释，在该模型中肽分配到磷脂头部基团区域并沿膜/水界面扩散。如果膜中存在POPG，肽的碱性残基与酸性脂质头部基团之间的静电吸引力会导致肽更深入地穿透双层膜。对于这种情况，讨论了两个与实验数据都一致的模型，在其中肽要么形成由三到六个部分螺旋的跨膜单体组成的寡聚体，要么以其两亲性螺旋段平行于膜平面排列并位于双层膜的头部基团和外部烃区域附近的方式插入双层膜中。
