Swamy M J, Marsh D
Abteilung Spektroskopie, Max-Planck-Institut für biophysikalische Chemie, 37070 Göttingen, Germany.
Biochemistry. 2001 Dec 11;40(49):14869-77. doi: 10.1021/bi0029189.
The interaction of avidin with aqueous dispersions of N-biotinylphosphatidylethanolamines, of acyl chain lengths C(14:0), C(16:0), and C(18:0), was studied by using spin-label electron spin resonance (ESR) spectroscopy, (31)P nuclear magnetic resonance ((31)P NMR) spectroscopy, differential scanning calorimetry, and chemical binding assays. In neutral buffer containing 1 M NaCl, binding of avidin is due to specific interaction with the biotinyl lipid headgroup because avidin presaturated with biotin does not bind. Saturation binding of the protein corresponds to a ratio of 50 lipid molecules per tetrameric avidin. Phospholipid probes spin-labeled at various positions between C-4 and C-14 in the sn-2 chain were used to characterize the effects of avidin binding on the lipid chain dynamics. In the fluid phase, protein binding results in a decrease of chain mobility at all positions of labeling while the flexibility gradient characteristic of a liquid-crystalline lipid phase is maintained. There is no evidence from the spin-label ESR spectra for penetration of the protein into the hydrophobic interior of the membrane. At temperatures corresponding to the gel phase, the lipid chain mobility increases on binding protein. The near constancy in mobility found with chain position, however, suggests that in the gel phase the lipid chains remain interdigitated upon binding avidin. Binding of increasing amounts of avidin results in a gradual decrease of the lipid chain-melting transition enthalpy with only small change in the transition temperature. At saturation binding, the calorimetric enthalpy is reduced to zero. (31)P NMR spectroscopy indicates that protein binding increases the surface curvature of dispersions of all three biotin lipids. The C(14:0) biotin lipid yields isotropic (31)P NMR spectra in the presence of avidin at all temperatures between 10 and 70 degrees C, in contrast to dispersions of the lipid alone, which give lamellar spectra at low temperature that become isotropic at the chain-melting temperature. In the presence of avidin, the C(16:0) and C(18:0) biotin lipids yield primarily lamellar (31)P NMR spectra at low temperature with a small isotropic component; the intensity of the isotropic component increases with temperature, and the spectra narrow and become totally isotropic at high temperature, in contrast to dispersions of the lipids alone, which give lamellar spectra in the fluid phase. The binding of avidin therefore reduces the cooperativity of the biotin lipid packing, regulates the mobility of the lipid chains, and enhances the surface curvature of the lipid aggregates. These effects may be important for both lateral and transbilayer communication in the membrane.
通过使用自旋标记电子自旋共振（ESR）光谱、磷-31核磁共振（³¹P NMR）光谱、差示扫描量热法和化学结合测定法，研究了抗生物素蛋白与酰基链长度分别为C(14:0)、C(16:0)和C(18:0)的N-生物素磷脂酰乙醇胺水分散体之间的相互作用。在含有1 M NaCl的中性缓冲液中，抗生物素蛋白的结合是由于与生物素脂质头部基团的特异性相互作用，因为预先用生物素饱和的抗生物素蛋白不会结合。蛋白质的饱和结合对应于每个四聚体抗生物素蛋白与50个脂质分子的比例。使用在sn-2链中C-4至C-14之间的不同位置进行自旋标记的磷脂探针来表征抗生物素蛋白结合对脂质链动力学的影响。在流体相中，蛋白质结合导致标记所有位置的链流动性降低，同时保持液晶脂质相特有的柔韧性梯度。自旋标记ESR光谱没有证据表明蛋白质渗透到膜的疏水内部。在对应于凝胶相的温度下，结合蛋白质后脂质链的流动性增加。然而，链位置处流动性的近乎恒定表明，在凝胶相中，结合抗生物素蛋白后脂质链仍保持相互交错。增加抗生物素蛋白的结合量会导致脂质链熔化转变焓逐渐降低，而转变温度只有很小的变化。在饱和结合时，量热焓降至零。³¹P NMR光谱表明，蛋白质结合增加了所有三种生物素脂质分散体的表面曲率。与单独脂质的分散体不同，单独脂质的分散体在低温下给出层状光谱，在链熔化温度下变为各向同性，而C(14:0)生物素脂质在10至70摄氏度之间的所有温度下，在抗生物素蛋白存在下产生各向同性的³¹P NMR光谱。在抗生物素蛋白存在下，C(16:0)和C(18:0)生物素脂质在低温下主要产生层状³¹P NMR光谱，有少量各向同性成分；各向同性成分的强度随温度增加，光谱变窄并在高温下变为完全各向同性，而单独脂质的分散体在流体相中给出层状光谱。因此，抗生物素蛋白的结合降低了生物素脂质堆积的协同性，调节了脂质链的流动性，并增强了脂质聚集体的表面曲率。这些效应可能对膜中的横向和跨膜通讯都很重要。
