Dentino A R, Westerman P W, Yeagle P L
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo 14214, USA.
Biochim Biophys Acta. 1995 May 4;1235(2):213-20. doi: 10.1016/0005-2736(95)80007-3.
The anti-viral and membrane fusion inhibitor, carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine (ZfFG), was studied in phospholipid bilayers, where earlier studies had indicated this peptide functioned. Multinuclear magnetic resonance (NMR) studies were performed with isotopically labeled peptide. A peptide labeled in the glycine carboxyl with 13C was synthesized, and the isotropic 13C-NMR chemical shift of that carbon was measured as a function of pH. A pKa of 3.6 for the carboxyl was determined from the peptide bound to a phosphatidylcholine bilayer. ZfFG inhibits the formation by sonication of highly curved, small unilamellar vesicles. Experiments as a function of pH revealed that this ability of ZfFG was governed by a pKa of 3.7. Therefore the protonation state of the carboxyl of ZfFG appeared to regulate the effectiveness of this anti-viral peptide at destabilizing highly curved phospholipid assemblies. Such destabilization had previously been discovered to be related to the mechanism of the anti-fusion and anti-viral activity of this peptide. The location of the carboxyl of ZfFG in the membrane was probed with paramagnetic relaxation enhancement of the 13C spin lattice relaxation of the carboxyl carbon in the glycine of ZfFG (enriched in 13C). Results suggested that this carboxyl is at or above the surface of the phospholipid bilayer. The dynamics of the molecule in the membrane were examined with 2H-NMR studies of ZfFG, deuterated in the alpha-carbon protons of the glycine. When ZfFG was bound to membranes of phosphatidylcholine, a sharp 2H-NMR spectral component was observed, consistent with a disordering of the glycine methylene segment of the peptide. When ZfFG was bound to N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE) bilayers at temperatures below 30 degrees C, a large quadrupole splitting was observed. These results suggest that ZfFG likely inhibits membrane fusion from the surface of the lipid bilayer, but not by forming a tight, stoichiometric complex with the phospholipids.
抗病毒和膜融合抑制剂苄氧羰基-D-苯丙氨酸-L-苯丙氨酸-甘氨酸（ZfFG）在磷脂双层中进行了研究，早期研究表明该肽在其中发挥作用。使用同位素标记的肽进行了多核磁共振（NMR）研究。合成了在甘氨酸羧基用13C标记的肽，并测量了该碳的各向同性13C-NMR化学位移作为pH的函数。从与磷脂酰胆碱双层结合的肽中确定羧基的pKa为3.6。ZfFG抑制通过超声处理形成高度弯曲的小单层囊泡。作为pH函数的实验表明，ZfFG的这种能力受pKa 3.7的控制。因此，ZfFG羧基的质子化状态似乎调节了这种抗病毒肽在破坏高度弯曲的磷脂组装体时的有效性。此前已发现这种破坏与该肽的抗融合和抗病毒活性机制有关。用顺磁弛豫增强ZfFG（富含13C）甘氨酸中羧基碳的13C自旋晶格弛豫来探测ZfFG羧基在膜中的位置。结果表明该羧基位于磷脂双层表面或之上。用ZfFG在甘氨酸的α-碳质子中氘代的2H-NMR研究检查了分子在膜中的动力学。当ZfFG与磷脂酰胆碱膜结合时，观察到一个尖锐的2H-NMR光谱成分，这与肽的甘氨酸亚甲基片段的无序化一致。当ZfFG在低于30摄氏度的温度下与N-甲基二油酰磷脂酰乙醇胺（N-甲基DOPE）双层结合时，观察到较大的四极分裂。这些结果表明，ZfFG可能从脂质双层表面抑制膜融合，但不是通过与磷脂形成紧密的化学计量复合物来实现。
