Secondary structure of bacteriorhodopsin fragments. External sequence constraints specify the conformation of transmembrane helices.
The secondary structure of bacteriorhodopsin polypeptides comprising two (AB, CD, DE, FG), three (AC, CE, EG), four (AD, DG), or five (AE, CG) of the seven transmembrane segments has been analyzed by circular dichroism spectroscopy. A comparison of the alpha-helix content with that predicted from the high resolution structure of the native protein revealed that the N-terminal AB, AC, AD, and AE fragments and the C-terminal CG fragment are completely refolded in the presence of mixed phospholipid micelles. In contrast, the DG, EG, FG, CD, CE, and DE fragments did not form alpha-helices of the expected lengths at pH 6. Each of the latter fragments displayed, however, an increased helicity upon lowering the pH to 4. Fluorescence measurements with the CD and FG fragments suggest that this helix formation occurs within transmembrane segments C and G, respectively, and thus is likely to originate from the protonation of carboxyl residues that participate in proton translocation. The partial misfolding at neutral pH observed for the shorter fragments from the central and C-terminal part of bacteriorhodopsin indicates that the conformation of some transmembrane segments is specified by interactions with neighboring helices in the assembled structure. Moreover, the data demonstrate that two stable helices at the N terminus of a multihelical membrane protein are sufficient as a folding template to induce a native conformation to the following transmembrane domains.
Lüneberg
J
J
Department of Molecular Biology, Bernhard Nocht Institute, D-20359 Hamburg, Germany.
Widmann
M
M
Dathe
M
M
Marti
T
T
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D010446:Peptide Fragments; D001436:Bacteriorhodopsins