Chang D K, Chien W J, Arunkumar A I
Institute of Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China.
Biophys J. 1997 Feb;72(2 Pt 1):554-66. doi: 10.1016/s0006-3495(97)78695-8.
A peptide corresponding to the neuronal protein neurogranin (NG) residues 28-43, NG(28-43), and its analog, [A35]NG(28-43), have been investigated by NMR, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies. The peptides existed in aqueous solution predominantly in random form. However, a nascent helical structure was detected in the central region of the parent peptide from NMR data. Furthermore, a helical structure can be detected for both peptides with greater induced secondary structure for the parent peptide in the presence of sodium dodecyl sulfate (SDS) micelle. The formation of micelles for SDS was confirmed by results from EPR as well as 13C NMR. As shown by CD experiments, helical conformer was induced for NG(28-43) in vesicular solution containing phosphatidyl serine (PS), whereas no helix can be discerned for the peptide in phosphatidyl choline (PC)-containing vesicular solution. Together with the induction of the peptide into helix in SDS micellar solution as suggested by both NMR and CD data, these results underscored the electrostatic contribution to the interaction of the PKC substrate peptides and proteins with membrane. According to NMR and CD data, a dynamic equilibrium existed between free and micelle-bound states for the peptide. Moreover, proton-deuterium exchange results and SDS-induced linewidth broadening of proton resonances allowed delineation of the orientation of the amphipathic helix on the surface of SDS micelle. The result was supported by spin label experiments that indicated F35 of NG(28-43) interacted strongly with the hydrocarbon interior of micelle. Based on the experimental findings, a working model was proposed that attempted to partly explain the roles played by the nonpolar amino acid near the phosphorylation site, by the negatively charged phospholipids, and by the basic amino acids of the substrate.
一种对应于神经元蛋白神经颗粒素（NG）第28 - 43位残基的肽段，即NG(28 - 43)，及其类似物[A35]NG(28 - 43)，已通过核磁共振（NMR）、电子顺磁共振（EPR）和圆二色性（CD）光谱进行了研究。这些肽段在水溶液中主要以无规形式存在。然而，根据NMR数据，在母体肽段的中心区域检测到了一种新生的螺旋结构。此外，在十二烷基硫酸钠（SDS）胶束存在的情况下，两种肽段都能检测到螺旋结构，且母体肽段具有更大的诱导二级结构。EPR以及13C NMR的结果证实了SDS胶束的形成。如CD实验所示，在含有磷脂酰丝氨酸（PS）的囊泡溶液中，NG(28 - 43)诱导形成了螺旋构象，而在含有磷脂酰胆碱（PC）的囊泡溶液中该肽段未观察到螺旋结构。NMR和CD数据均表明该肽段在SDS胶束溶液中诱导形成螺旋结构，这些结果强调了静电作用对蛋白激酶C（PKC）底物肽段和蛋白质与膜相互作用的贡献。根据NMR和CD数据，该肽段在游离态和胶束结合态之间存在动态平衡。此外，质子 - 氘交换结果以及SDS诱导的质子共振线宽展宽使得能够描绘两亲性螺旋在SDS胶束表面的取向。自旋标记实验支持了这一结果，表明NG(28 - 43)的F35与胶束的烃类内部强烈相互作用。基于实验结果，提出了一个工作模型，试图部分解释磷酸化位点附近的非极性氨基酸、带负电荷的磷脂以及底物的碱性氨基酸所起的作用。
