Optical Activity of Biological Membranes: Scattering Effects and Protein Conformation

Abstract

Abstract. The following question has recently arisen in the literature concerning the interpretation of the optical activity of biological membranes: do the characteristic spectral distortions observed for diverse membrane systems reflect some common and unique aspect of membrane architecture or are they the result of scattering effects owing to the particulate nature of membranous systems? We have confirmed the latter interpretation on the basis of the following experimental observations: (a) red blood cell membranes give a normal circular dichroism spectrum when scattering is reduced and (b) nonaggregated, nonmembranous helical proteins give distorted membranelike spectra when scattering is introduced. An improved estimate of secondary structure on the basis of undistorted spectra results in about 50 per cent a-helix for red blood cell membrane protein. In addition we conclude that the distortions in optical activity spectra offer no evidence in support of various proposed membrane models.During recent years there has arisen a literature concerning the optical rotatory dispersion and circular dichroism of biological membranes.1-9 Optical activity is a sensitive measure of the secondary structure of proteins and offers an attractive nondestructive probe of intact membrane structure. Many different membrane systems have given very similar optical rotatory dispersion and circular dichroism spectra, resembling an a-helical protein, but with characteristic distortions in relative intensity of the 208 nm 7r-r* to 222 nm n-7r* troughs, and red shifts in the n-7r* trough and crossover wavelengths. Several interpretations have been offered to account for these distortions in the membrane spectra. It was first proposed that since this unique spectra occurs in diverse membrane systems, it must represent some common aspect of membrane architecture, e.g., protein aggregation with possible alignment of a-helicesl 2 or hydrophobic interaction between protein helices and lipid.3 On the other hand, there has been the recent proposal of Urry and Ji8'9 that the distortions in the membrane spectra are the result of scattering artifacts owing to the particulate nature of the system. There is some evidence correlating turbidity with spectral distortions,2'9 but most of these experiments do not distinguish between effects resulting from scattering and those resulting from aggregation-induced polypeptide interactions. Ji and Urry's9 results on sonicated mitochondrial membrane fractions are the most convincing. However, since mitochondrial membranes are notably heterogeneous and a spectrum of the total sonicate was not 793

Optical Activity of Membrane Suspensions: Calculation of Artifacts by Mie Scattering Theory

“…The two artifacts oppose each other in the region X <200 nm, s0 that the solution and suspension differ little in the amplitude of their positive CD band in this region. It has been shown experimentally that sonication of ghost suspensions generates small solvent-filled vesicles for which the CD anomalies are much reduced (8). In Fig.…”

“…Ji and Urry, (6) were the first to present experimental evidence that these anomalies might instead be artifactual, when they showed that suspensions of poly(iglutamic acid) [(Glu)n] aggregates exhibit the same red shift and distortion in their CD spectra. The experiments of Schneider et al (8), which demonstrate both the diminution of these anomalies upon sonication of erythrocyte ghosts and their enhancement in the CD of partially hemolyzed erythrocytes, provide further convincing experimental evidence for a significant artifactual component in the optical activity of membranes. There have also been some attempts to develop a quantitative theory to account for the observed optical activity of membranes (9)(10)(11)(12)(13)(14); none of these has been truly satisfactory.…”

The circular dichroism of bacteriorhodopsin: Asymmetry and light‐scattering distortions

“…In the analysis of the CD-spectra of suspensions of large particles distorting effects due to light-scattering, on band shape, as well as intensity, should be taken into consideration [2][3][4]. It is shown in this work both experimentally and theoretically that the asymmetry in the CD-spectrum of the purple membrane in water suspensions can be attributed to such scattering distortions.…”

Membrane-promoted unfolding of acetylcholinesterase: A possible mechanism for insertion into the lipid bilayer

“…3), showed a reduced ellipticity of the bound protein, as compared with native AcChoEase, but greater ellipticity than for AcChoEase in a fully unfolded (U) state in 5 M Gdn⅐HCl, as would be expected for AcChoEase in an MG state (24). It is well known that CD spectroscopy of membrane preparations must take into account possible scattering artifacts (25,26). This precluded accurate CD measurements in the near UV, where the contribution of the aromatic bands produces relatively low ellipticity.…”