Department of Physics, University of Calabria, Ponte P. Bucci 31C, 87036 Rende (CS), Italy.
J Phys Chem B. 2012 Sep 27;116(38):11608-15. doi: 10.1021/jp3074392. Epub 2012 Sep 18.
β-Lactoglobulin (β-LG) is a member of the lipocalin protein family involved in the transport of fatty acids and other small hydrophobic molecules. The main binding site is at a central cavity, referred to as "calyx", formed by the protein β-barrel sandwich. Continuous-wave and pulsed Fourier transform electron spin resonance (cw- and FT-EPR) spectroscopy and molecular dynamics (MD) simulation were combined to investigate the interaction of fatty acids with bovine β-LG. Stearic acid bearing the nitroxide label at different positions, n, along the acyl chain (n-SASL, n = 5, 7, 10, 12, 16) were used. The EPR data show that the protein affinity for SASL decreases on going from n = 5 to 16. This behavior is due to the accommodation of the SASL in the protein calyx, which is hampered by steric hindrance of the doxyl ring for n ≥ 10, as evidenced by MD data. Conformation and dynamics of 5-SASL are similar to those of the unlabeled stearate molecule. 5-SASL in the protein binding site undergoes librational motion of small amplitude on the nanosecond time scale at cryogenic temperature and rotational dynamics with correlation time of 4.2 ns at physiological temperature. The results highlight the dynamical features of fatty acids/β-LG interaction.
β-乳球蛋白（β-LG）是一种参与脂肪酸和其他小疏水分子运输的脂联素蛋白家族成员。主要结合位点位于由蛋白质β-桶夹心形成的中央腔中，称为“杯状结构”。连续波和脉冲傅里叶变换电子自旋共振（cw-和 FT-EPR）光谱和分子动力学（MD）模拟相结合，研究了脂肪酸与牛β-LG 的相互作用。使用了带有不同位置氮氧自由基标记的硬脂酸，酰链上的 n（n-SASL，n = 5、7、10、12、16）。EPR 数据表明，SASL 与蛋白质的亲和力随 n 从 5 增加到 16 而降低。这种行为归因于 SASL 在蛋白质杯状结构中的容纳，这受到 doxyl 环的空间位阻的阻碍，MD 数据证明了这一点。5-SASL 的构象和动力学与未标记的硬脂酸分子相似。在低温下，蛋白质结合位点中的 5-SASL 会在纳秒时间尺度上进行小振幅的摆动运动，在生理温度下，其旋转动力学的相关时间为 4.2ns。这些结果突出了脂肪酸/β-LG 相互作用的动态特征。
