Abstract
Bacteriorhodopsin is a heptahelical membrane protein that can be refolded to the native state following denaturation. To analyze the in vitro folding process with independent structural domains, eight fragments comprising two (AB, FG), three (AC, EG), four (AD, DG) or five (AE, CG) of the transmembrane segments were produced by expression in Escherichia coli. The polypeptides were purified to homogeneity by solvent extraction of E. coli membranes, repeated phase separation, and anion-exchange chromatography employing the C-terminal tail of bacteriorhodopsin for adsorption. Upon reconstitution into phospholipid/detergent micelles pairs of complementary fragments (AB.CG, AC.DG, AD.EG, and AE.FG) assembled in the presence of retinal to regenerate the characteristic bacteriorhodopsin chromophore with high efficiency. Together with previous studies, these results demonstrate that the covalent connections in each of the six interhelical loops are dispensable for a correct association of the helices. The different loops, however, contribute to the stability of the folded structure, as shown by increased susceptibilities toward denaturation in SDS and at acidic pH, and decreased Schiff base pKa values for the AB.CG, AC. DG, AD.EG, and AE.FG complexes, compared with the intact protein. Notably, the heptahelical bundle structure was also generated by all possible combinations of pairs of overlapping fragments, containing one (AC.CG, AD.DG, AE.EG), two (AD.CG, AE.DG), or three (AE.CG) redundant helices. The spectral properties of the chromophores indicate that the retinal-binding pocket of the AC.CG, AD.CG, and AE. CG complexes is formed by helices A and B of the respective N-terminal fragment and the C-terminal CG fragment, whereas the AD. DG, AE.DG, and AE.EG complexes are likely to adopt a heptahelical bundle structure analogous to AD.EG. The combined data show that the specificity of the helix assembly of bacteriorhodopsin is influenced by connectivities provided by the C-D and E-F surface loops.
Highlights
Bacteriorhodopsin (BR)1 is an integral membrane receptor that functions as a light-driven proton pump in the purple membrane of Halobacterium salinarium [1,2,3,4]
The large degree of structural autonomy of membrane-spanning polypeptides is supported by experiments, in which native structures have been assembled from fragments that were separately refolded in vitro or synthesized in vivo
Whereas chromophore formation of eBR at 22 °C was dominated by the fast kinetic component that accounted for 85% of the absorbance change, the two kinetic components contributed more or less to the refolding process of the fragment complexes (Table III)
Summary
(Received for publication, December 17, 1997, and in revised form, January 27, 1998). Upon reconstitution into phospholipid/detergent micelles, all pairs of fragments comprising complementary (AB1⁄7CG, AC1⁄7DG, AD1⁄7EG, AE1⁄7FG) or overlapping (AC1⁄7CG, AD1⁄7CG, AD1⁄7DG, AE1⁄7CG, AE1⁄7DG, AE1⁄7EG) parts of the protein regenerated a native-like chromophore with high efficiency, whereas in the absence of one or more of the transmembrane segments pigment formation was abolished. This shows that the assembly of BR requires each of the seven helices but does not depend on any of the covalent connections provided by the surface loops.
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