Han X, Li G, Li G, Lin K
Department of Biophysics, Beijing Medical University, P. R. China.
Biochemistry. 1998 Jul 28;37(30):10730-7. doi: 10.1021/bi9800451.
Fourier transform infrared (FTIR) spectroscopy has been carried out to investigate the thermal denaturation of alpha-actinin and its complexes with dioleoylphosphatidylglycerol (DOPG) vesicles. The amide I regions in the deconvolved spectra of alpha-actinin in the lipid-free and DOPG-bound states are both consistent with predominantly alpha-helical secondary structure below the denaturation temperatures. Studies of the temperature dependence of the spectra revealed that for alpha-actinin alone the secondary structure was unaltered up to 40 degrees C. But, in the presence of DOPG vesicles, the thermal stability of the secondary structure of alpha-actinin increased to 55 degrees C. The thermal denaturation mechanisms of the lipid-free and DOPG-bound states of alpha-actinin also vary. The secondary structure of the lipid-free alpha-actinin changed to be predominantly unordered upon heating to 65 degrees C and above. Whereas, the original alpha-helical structure in the DOPG-bound alpha-actinin retained even at 70 degrees C, the highest temperature we examined. Analysis of the reduction in amide II intensities, which is due to peptide H-D exchange upon heating alpha-actinin in D2O, showed that partially unfolded states with increased solvent accessibility but substantial secondary structures could be observed from 35 to 40 degrees C only if DOPG vesicles were present. A so-called "protamine precipitation" method has been developed to purify the N-terminal domain of alpha-actinin by use of the fact that the central domain of alpha-actinin is negatively charged but the N-terminal domain is positively charged. Thermal denaturation of the central and N-terminal domains of alpha-actinin were then investigated with FTIR. The secondary structure of the N-terminal domain of alpha-actinin was found to be thermally sensitive below 35 degrees C, which is characterized as the increase of the alpha-helical structure at the expense of the random coil upon heating the N-terminal domain from 4 to 35 degrees C. The membrane-binding ability of the N-terminal domain of alpha-actinin was proposed in terms of the analysis of the local electrostatic properties of alpha-actinin and the assignment of the amide II bands in the FTIR spctra of alpha-actinin.
已进行傅里叶变换红外（FTIR）光谱分析，以研究α-辅肌动蛋白及其与二油酰磷脂酰甘油（DOPG）囊泡形成的复合物的热变性。在无脂质和结合DOPG状态下，α-辅肌动蛋白解卷积光谱中的酰胺I区域均与变性温度以下主要为α-螺旋二级结构一致。对光谱温度依赖性的研究表明，单独的α-辅肌动蛋白在40℃以下二级结构未改变。但是，在存在DOPG囊泡的情况下，α-辅肌动蛋白二级结构的热稳定性提高到55℃。α-辅肌动蛋白无脂质和结合DOPG状态的热变性机制也有所不同。无脂质的α-辅肌动蛋白加热到65℃及以上时，二级结构主要变为无序状态。而结合DOPG的α-辅肌动蛋白即使在我们检测的最高温度70℃时仍保留原始的α-螺旋结构。对酰胺II强度降低的分析表明，这是由于在重水中加热α-辅肌动蛋白时肽的H-D交换所致，结果显示仅在存在DOPG囊泡的情况下，从35℃到40℃可观察到溶剂可及性增加但具有大量二级结构的部分解折叠状态。已开发出一种所谓的“鱼精蛋白沉淀”方法，利用α-辅肌动蛋白的中央结构域带负电荷而N端结构域带正电荷这一事实来纯化α-辅肌动蛋白的N端结构域。然后用FTIR研究α-辅肌动蛋白中央和N端结构域的热变性。发现α-辅肌动蛋白N端结构域的二级结构在35℃以下对热敏感，其特征是在将N端结构域从第4℃加热到35℃时，α-螺旋结构增加，以无规卷曲为代价。根据对α-辅肌动蛋白局部静电性质的分析以及α-辅肌动蛋白FTIR光谱中酰胺II带的归属，提出了α-辅肌动蛋白N端结构域的膜结合能力。
