Abstract
The secondary structure of bacterio-opsin (BO), the retinal free protein-component of bacteriorhodopsin (BR), has been determined by Raman spectroscopy. Additional circular dichroism (CD) measurements have revealed only negligible conformational differences between BO in apomembranes and BR in purple membranes. Therefore, the secondary structure of BR was derived from the Raman data of BO. The protein conformation was determined to consist of 72-82% helices, 2-11% beta-strands, and 11-17% beta-turns. Only about half of the helical structures correspond to alpha 1-helices, the other half possess non-alpha 1-helical structures. According to the analysis of the Raman data, the derived secondary structure of BR was obtained with high reliability for all structure classes which can be distinguished by this method within the given uncertainty range. This is a remarkable difference from recently published secondary structural data derived from CD measurements where the helix content was reported to be between 50 and 80%. The inherent experimental and methodological uncertainties of the CD-technique leading to such a range of variation are critically discussed in comparison to the method of Raman spectroscopy. The combined application of Raman and CD spectroscopy, as performed here, is demonstrated to be a substantial improvement in the secondary structure determination of retinal-containing membrane proteins. On the basis of our results, some of the recently proposed structural models of BR with a beta-strand content of more than 11% can be ruled out.
Highlights
The secondary structure of bacteria-opsin (BO), the retinal free protein-component of bacteriorhodopsin (BR), has been determined by Raman spectroscopy
Published IR spectra of BR make it highly plausible that a distorted ahelical conformation is the predominant form of the polypeptide secondary structure, thereis no clear answer for the exact amount of thisstructural element (7, 8-12)
Besides the distorted a-helix form, which was interpreted as an aII-helix by normal mode calculations (13), IR spectra indicate that BR contains in addition an appreciable amount of 0-strands (7, 10, 11).Based on IR and CD measurements, Jap et al (7) proposed a new structural model of BR, where the apparentseven transbilayer segments seen in the electron microscopy areinterpreted to consist of five a-helices and four P-strands
Summary
From the $Biozentrum der Universitat Basel,Klingelbergstrasse 70, CH-4056 Basel, Switzerland and the Slnstitut f u r Bwbgie I (Zoologie) der Universitat Freiburg, Alberstrasse 214 0-78 Freiburg, Federal Republic of Germany. Secondary structural data obtained by CD are in general much less reliable than those determined by Raman spectroscopy, a conformational difference between two proteins will be reflected correctly in the CD spectra, at leastin the a-helix content.Takinginto account the analysis of the CD measurements, the secondary structure of BR was derived from the Raman datoaf BO. Light scattering effects in the absorption measurements were taken into consideration by using for base-line measurements an aqueous suspension of apomembranes of identical protein concentration as that of the purple membranes in the first case, and a micellar solution in 80 mM octyl glucoside in the latter case.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
