Mao D, Wallace B A
Biochemistry. 1984 Jun 5;23(12):2667-73. doi: 10.1021/bi00307a020.
The large size of membrane particles and the high local concentration of proteins in these particles give rise to differential scattering and absorption flattening effects which result in significant distortions of the circular dichroism spectra of membrane proteins and produce erroneous estimates of secondary structure. In an attempt to find a membrane system in which scattering and flattening are minimal, but in which native protein conformation is retained, several methods of fragmentation, including sonication, solubilization, and incorporation into small unilamellar vesicles (SUVs), were examined. Bacteriorhodopsin in purple membrane sheets was used as a test system for the effectiveness of the procedures since its secondary structure is known from independent physical measurements and these large membranes produce considerable distortions, as seen by comparison of observed and calculated spectra for the protein. While sonication decreased differential scattering, it had little effect on the total distortion; solubilization in octyl glucoside tended to decrease both differential scattering and flattening but induced some conformational change in the protein. However, when bacteriorhodopsin was incorporated into small unilamellar vesicles, which both decrease particle size and dilute the local concentration of protein, the spectrum produced was nearly identical with the calculated one, suggesting that SUVs may be appropriate vehicles for use with membrane proteins and may be a facile method for eliminating optical artifacts.
膜颗粒的大尺寸以及这些颗粒中蛋白质的高局部浓度会产生不同的散射和吸收扁平化效应，这会导致膜蛋白圆二色光谱的显著畸变，并产生二级结构的错误估计。为了找到一个散射和扁平化最小但能保留天然蛋白质构象的膜系统，研究了几种破碎方法，包括超声处理、增溶以及掺入小单层囊泡（SUVs）。紫色膜片中的细菌视紫红质被用作测试这些程序有效性的系统，因为其二级结构已通过独立的物理测量得知，并且这些大膜会产生相当大的畸变，通过比较该蛋白质的观察光谱和计算光谱可以看出。虽然超声处理减少了不同的散射，但对总畸变影响不大；在辛基葡糖苷中增溶往往会减少不同的散射和扁平化，但会引起蛋白质的一些构象变化。然而，当细菌视紫红质掺入小单层囊泡时，这既减小了颗粒尺寸又稀释了蛋白质的局部浓度，产生的光谱与计算光谱几乎相同，这表明SUVs可能是用于膜蛋白的合适载体，并且可能是消除光学假象的简便方法。
