Georghiou S, Thompson M, Mukhopadhyay A K
Biochim Biophys Acta. 1982 Jun 14;688(2):441-52. doi: 10.1016/0005-2736(82)90355-8.
The rotational correlation time of melittin, obtained from the nanosecond anisotropy of the emission from its single tryptophan residue, has been found to increase considerably in phosphate solution relative to that in aqueous solution, consistent with protein aggregation. The steady-state fluorescence spectra as well as the absorption spectra in phosphate solution exhibit a very good degree of similarity with those of the protein bound to egg phosphatidylcholine (PC) and distearoylphosphatidylcholine (DSPC) bilayer liposomes. The value of the second-order rate constant for dynamic quenching, kq = 1.4.10(9) M-1.s-1, by acrylamide in 0.5 M phosphate solution is comparable to those for the protein-phospholipids complexes (1.10(9) and 0.7.10(9) M-1.s-1 for egg PC and DSPC, respectively). Similarities are also found in the nanosecond properties. There is a much stronger and quite similar dependence of the fluorescence spectra on time in the nanosecond range and of the fluorescence decay times on the emission wavelength in both cases as compared to the case is aqueous solution. These observations support the notion that melittin binds to the phospholipids in an aggregated form. The results suggest that the reduction in the kq values of bound melittin relative to that in aqueous solution and the blue shift of the fluorescence spectrum (from 352 to 337 nm) are brought about by shielding of the tryptophan residue from the solvent through a combination of protein aggregation and enhancement of its alpha-helical content (suggested by published CD data). The magnitude of the kq values for bound melittin, however, is still relatively high implying the occurrence of rather frequent encounters between the tryptophan residue and the hydrophilic acrylamide molecules. Thus, the residue is found not to penetrate deep into the phospholipid bilayer.
通过蜂毒肽单个色氨酸残基发射光的纳秒级各向异性得到的蜂毒肽旋转相关时间，已发现其在磷酸盐溶液中相对于水溶液有显著增加，这与蛋白质聚集一致。磷酸盐溶液中的稳态荧光光谱以及吸收光谱与结合到卵磷脂（PC）和二硬脂酰磷脂酰胆碱（DSPC）双层脂质体上的蛋白质的光谱表现出非常高的相似性。在0.5 M磷酸盐溶液中，丙烯酰胺对蜂毒肽的动态猝灭二级速率常数kq = 1.4×10⁹ M⁻¹·s⁻¹，与蛋白质 - 磷脂复合物的该常数相当（卵磷脂PC和二硬脂酰磷脂酰胆碱DSPC分别为1×10⁹和0.7×10⁹ M⁻¹·s⁻¹）。在纳秒级特性方面也发现了相似之处。与水溶液情况相比，在这两种情况下，荧光光谱在纳秒范围内对时间的依赖性以及荧光衰减时间对发射波长的依赖性都要强得多且非常相似。这些观察结果支持了蜂毒肽以聚集形式与磷脂结合的观点。结果表明，与水溶液相比，结合态蜂毒肽kq值的降低以及荧光光谱的蓝移（从352 nm到337 nm）是由于蛋白质聚集和α - 螺旋含量增加（已发表的圆二色性数据表明）对色氨酸残基进行溶剂屏蔽所致。然而，结合态蜂毒肽的kq值仍然相对较高，这意味着色氨酸残基与亲水性丙烯酰胺分子之间相当频繁地发生碰撞。因此，发现该残基并未深入穿透磷脂双层。
