Refolding of bacteriorhodopsin from expressed polypeptide fragments.
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.
Marti
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Department of Molecular Biology, Bernhard Nocht Institute, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany. marti@bni.uni-hamburg.de
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003624:Darkness; D003902:Detergents; D004926:Escherichia coli; D005798:Genes, Bacterial; D008027:Light; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012997:Solvents; D013053:Spectrophotometry
D003902:Detergents; D008823:Micelles; D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins; D012997:Solvents; D001436:Bacteriorhodopsins