Tertiary structure of bacteriorhodopsin. Positions and orientations of helices A and B in the structural map determined by neutron diffraction.
Positions and rotations of two helices in the tertiary structure of bacteriorhodopsin have been studied by neutron diffraction using reconstituted, hybrid purple membrane samples. Purple membrane was biosynthetically 2H-labeled at non-exchangeable hydrogen positions of leucine and tryptophan residues. Two chymotryptic fragments were purified, encompassing either the first two or the last five of the seven putative transmembrane segments identified in the amino acid sequence of bacteriorhodopsin. The 2H-labeled fragments, diluted to variable extents with the identical, unlabeled fragment, were mixed with their unlabeled counterpart; bacteriorhodopsin was then renatured and reconstituted. The crystalline purple membrane samples thus obtained contained hybrid bacteriorhodopsin molecules in which certain transmembrane segments had been selectively 2H-labeled to various degrees. Neutron diffraction powder patterns were recorded and analyzed both by calculating difference Fourier maps and by model building. The two analyses yielded consistent results. The first and second transmembrane segments in the sequence correspond to helices 1 and 7 of the three-dimensional structure, respectively. Rotational orientations of these two helices were identified using best fits to the observed diffraction intensities. The data also put restrictions on the position of the third transmembrane segment. These observations are discussed in the context of folding models for bacteriorhodopsin, the environment of the retinal Schiff base, and site-directed mutagenesis experiments.
Popot
J L
JL
Institut de Biologie Physico-Chimique, Paris, France.
Engelman
D M
DM
Gurel
O
O
Zaccaï
G
G
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000648:ultrastructure*; D003196:Computer Graphics; D005583:Fourier Analysis; D006217:Halobacterium; D008565:Membrane Proteins / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D009502:Neutrons; D011487:Protein Conformation; D012542:Scattering, Radiation
D008565:Membrane Proteins; D001436:Bacteriorhodopsins