THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc Vol. 266, No. 13, Issue of May 5, pp. 8545-8550.1991 Printed in U. S. A. Structure-Function Studies of Bacteriorhodopsin XV EFFECTS OF DELETIONS IN LOOPS B-C AND E-F ON BACTERIORHODOPSIN CHROMOPHORE AND STRUCTURE* (Received for publication, November 15, 1990) Marie A. Gilles-GonzalezSQ, Donald M. Engelmann, and H. Gobind KhoranaS From the $Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and the TDepartment of Molecular Biophysics and Biochemistry, Yale university, New Haven, Connecticut 0651 1 Bacteriorhodopsin mutants containing deletions in loop B-C, AT~P-G~u~~ or AGly65-Gln75, or a deletion in the loop E-F, AG1u'61-Ala166, were prepared. Fol- lowing their expression in Escherichia coli, the mutant proteins were purified to homogeneity and refolded with retinal in detergent-phospholipid mixtures. The mutants containing deletions in the loop B-C were normal at 4 "C but showed the following changes at 20 "C. 1) The X,,, shifted from 540 to below 510 nm; 2) the rates of bleaching by hydroxylamine in the dark increased; and 3) the rate and steady state of proton pumping decreased. Deletion of the eight amino acids in loop E-F did not affect wild-type behavior. However, all the mutant proteins were more prone to thermal and sodium dodecyl sulfate denaturation than the wild- type bacteriorhodopsin. These observations show that the structures of the B-C and E-F loops are not essential for correct folding of bacteriorhodopsin, but they con- tribute to the stability of the folded protein. Bacteriorhodopsin (bR),l an integral membrane protein in Halobacterium halobium, serves as a light-dependent proton pump (1). It consists of a single polypeptide chain of 248 amino acids and contains the chromophore all-tram retinal, linked via a Schiff base to lysine 216 (2-5). The model shown in Fig. 1 is based mainly on electron microscopy, with contri- butions from neutron diffraction, proteolysis studies, and photoaffinity labeling (9). Some uncertainty remains regard- ing the sizes of embedded helices and of the loops connecting the different helices. Loop B-C has been consistently large in * This work was supported by Grants GM28289 and AI 11479 from the National Institutes of Health and Grant N00014-82-K-0668 from the Office of Naval Research, Department of the Navy (to H. G. K.) and by National Science Foundation Grant DMB 8805587 and Na- tional Institutes of Health Grant 5 PO1 GM 22778 (to D. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Current address: University of California, San Diego, CA 92093. The abbreviations used are: bR, bacteriorhodopsin; hop, bacterio- opsin gene with native sequence; hop:lacZ, in-frame translational fusion of the nearly complete hop gene upstream to the E. coli lac2 gene, which yields a fusion protein with @-galactosidase activity; bo, bacterio-opsin; ebO, bacterio-opsin as obtained by expression of the hop gene and purified in vitro; ebR, bacteriorhodopsin obtained by regeneration of ebO in uitro; DMPC, L-a-dimyristoylphosphatidyl- choline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l-pro- panesulfonate; SDS, sodium dodecyl sulfate; aThr"-Gl~~~, a deletion of the eight ebO residues including threonine 67 to glutamate 74; AG1y6'-Gln7', a deletion of the eleven ebO residues including glycine 65 to glutamine 75; AG1u'fi1-Ala1fi8, a deletion of the eight ebO residues including glutamate 161 to alanine 168. various models proposed (6-9), and photoaffinity labeling experiments have indicated that E-F is also a large loop (7). In the present work we examine the effects of deletions in the loops on the folding, function, and stability of bR. Deletions of amino acids Thr'j-Gl~~~ and Gl~'j~-Gln~~ in the loop con- necting helices B and C were made and of amino acids G1ulfi1- AlaIfi8 in the loop between helices E and F. The B-C loop lies on the extracellular side of the membrane and the E-F loop on the cytoplasmic side. We found that all the deletions destabilize the structure to some extent, as measured by sensitivity to thermal and SDS denaturation. No effect was seen on proton pumping and the absorption spectrum in the case of the E-F deletion; however, the two deletions in loop B-C show blue shifts in the chro- mophore absorption maximum, diminished proton pumping, and increased susceptibility to hydroxylamine bleaching. In all cases, the basic folding of bR occurs as judged by circular dichroism, chromophore binding, and proton pumping. EXPERIMENTAL PROCEDURES Materials T4 DNA ligase was purified from Escherichia coli lysogenized with phage X expressing T4 ligase (10). Restriction endonucleases were from New England Biolabs and Boehringer Mannheim. The Klenow fragment of E. coli DNA polymerase I was purchased from Bethesda Research Laboratories. The "slow" form of nuclease Bal-31 was obtained from International Biotechnologies, Inc. Radiolabeled nu- cleotides were purchased from Amersham Corp. Nitrocellulose BA85 filter was from Schleicher and Schuell. Media for the growth of bacteria were supplied by Difco. DEAE-Trisacryl was obtained from LKB. Octyl glucoside and CHAPS were from Behring Diagnostics; DMPC was from Avanti Polar Lipids, Inc. Nucleotides, ampicillin, Nonidet P-40, phenylmethylsulfonyl fluoride, lysozyme, antifoam C, and RNase A were obtained from Sigma. All-trans retinal and 13-cis retinal were from Kodak. Methods General Recombinant DNA Methods-Digestions with restriction enzymes were performed as recommended by Fuchs and Blakesley (11) or by the commercial suppliers of the endonucleases. Both small- and large-scale plasmid preparations were done by alkaline sodium dodecyl sulfate lysis procedures (12, 13). In large-scale preparations, plasmid DNA was purified by equilibrium centrifugation in cesium chloride gradients containing ethidium bromide and by gel