Absorption and Optical Rotation Spectra of Biological Membranes

Abstract

The combination of ultraviolet absorption and circular dichroism (CD) spectroscopies provides a sensitive technique for following protein conformation and conformational changes in solution. It is reasonable to predict that the application of these methods to the study of biomembrane protein structure should yield valuable information about their averaged conformation and changes that might occur with changes in functional state. And indeed they do, after and only after appropriate data analyses, for the particulate nature of membranes gives rise to correctable scattering artifacts. In considering the CD spectrum of membranes, the objective is to eliminate the distortions due to their particulate nature by correcting the spectrum to the absorbance or difference absorbance values that would have occurred had the membrane proteins been molecularly dispersed in a state conformationally equivalent to that in the membrane. The corrected CD spectrum is then compared to reference spectra to estimate the average structure and/or compared to spectra of the membrane in different functional states to follow possible conformational changes. If a membrane suspension is challenged, for example, by the addition of substrate for one of the membrane-bound enzymes, and a difference in its CD spectrum is observed, this cannot be judged to be real until the scattering problem has been properly analyzed. This is not an onerous task, and indeed is straightforward, in large measure due to the arguments and methods known as the pseudoreference state (prs) approach described in detail in 1972(1) but whose origins go back to 1968. (2) This chapter will consider first absorption spectroscopy and then the related phenomenon of CD and its dispersion counterpart, optical rotatory dispersion, for both solutions and suspensions. The corrected spectra of the purple membrane provide a sample calculation demonstrating an application of the prs approach.