Differential Light Scattering and Absorption Flattening Optical Effects Are Minimal in the Circular Dichroism Spectra of Small Unilamellar Vesicles

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.