Stolowich N J, Frolov A, Atshaves B, Murphy E J, Jolly C A, Billheimer J T, Scott A I, Schroeder F
Department of Chemistry, Texas A&M University, College Station 77843-3255, USA.
Biochemistry. 1997 Feb 18;36(7):1719-29. doi: 10.1021/bi962317a.
The interaction and orientation of fatty acids with recombinant human sterol carrier protein-2 (SCP-2) were examined by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence techniques. 13C-NMR spectroscopy of stearic acid and oleic acid as well as fluorescence spectroscopy of cis-parinaric acid demonstrated that SCP-2 bound naturally occurring fatty acids with near 1:1 stoichiometry. Several findings indicated that the fatty acid was oriented in the binding site with its methyl end buried in the protein interior and its carboxylate exposed at the surface: the chemical shift of bound [18-13C]-stearate; dicarboxylic/monocarboxylic acid cis-parinaric acid displacement; complete ionization of the carboxylate group of SCP-2 bound [1-13C]stearate at neutral pH; lack of electrostatic interactions between 13C-fatty acids with SCP-2 cationic residues: pH titratability of the SCP-2 bound [1-13C]stearate carboxylate group. SCP-2 did not undergo global structural changes upon ligand binding or pH decrease as indicated by the absence of significant changes in NMR and only small alterations in time resolved fluorescence parameters. However, SCP-2 did undergo secondary structural changes detected by CD in the pH range 5-6. While these changes in secondary structure did not alter the fatty acid:SCP-2 binding stoichiometry, the affinity for fatty acid was increased severalfold at lower pH. In summary, 13C-NMR, CD, and fluorescence spectroscopy provided a detailed understanding of the interaction of fatty acids with SCP-2 and further showed for the first time the orientation of the fatty acid within the binding site. The pH-induced changes in SCP-2 secondary structure and ligand binding activity may be important to the mechanism whereby this protein interacts with membrane surfaces to enhance lipid binding/transfer.
通过核磁共振（NMR）、圆二色性（CD）和荧光技术研究了脂肪酸与重组人固醇载体蛋白-2（SCP-2）的相互作用及取向。硬脂酸和油酸的13C-NMR光谱以及顺式十八碳四烯酸的荧光光谱表明，SCP-2以接近1:1的化学计量比结合天然存在的脂肪酸。几项研究结果表明，脂肪酸在结合位点的取向是甲基端埋在蛋白质内部，羧基暴露在表面：结合的[18-13C]硬脂酸盐的化学位移；二羧酸/单羧酸顺式十八碳四烯酸置换；在中性pH下，与SCP-2结合的[1-13C]硬脂酸盐的羧基完全电离；13C-脂肪酸与SCP-2阳离子残基之间缺乏静电相互作用：与SCP-2结合的[1-13C]硬脂酸盐羧基的pH滴定性。如NMR无明显变化以及时间分辨荧光参数仅有微小改变所示，配体结合或pH降低时SCP-2未发生整体结构变化。然而，在pH值为5-6的范围内，CD检测到SCP-2确实发生了二级结构变化。虽然这些二级结构变化未改变脂肪酸与SCP-2的结合化学计量比，但在较低pH下对脂肪酸的亲和力增加了几倍。总之, 13C-NMR、CD和荧光光谱提供了对脂肪酸与SCP-2相互作用的详细理解，并首次进一步展示了脂肪酸在结合位点内的取向。pH诱导的SCP-2二级结构和配体结合活性的变化可能对该蛋白质与膜表面相互作用以增强脂质结合/转运的机制很重要。
