Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined.

Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of HI, 1H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor ru-st reprotonates the Schiffbase in the decay of M to N and is then reprotonated from the cytoplasm in the N -O 0 transition. The decay rate of N is proportional to [HI].By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (7)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like HI, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become >100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiffbase in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium halobium that transports H+ ions from the cytoplasm to the extracellular space with a stoichiometry of one proton per cycle (1). The transmembrane H+ translocation involves distinct electrogenic steps associated with H+ ejection from the protein interior into the periplasm and the subsequent H+ rebinding from the cytoplasmic side of the membrane. The proton uptake occurs on the millisecond time scale and is apparently coupled to the reprotonation of the Schiff base (SB) and the decay of the M intermediate of the photochemical cycle. Fourier transform infrared (FTIR) spectroscopy first indicated that several aspartate carboxyl groups undergo protonation changes during the photocycle (2, 3). Site-directed mutagenesis of bR showed that substitution of aspartate residues at positions 85, 96, and 212 by asparagine reduced the proton pumping activity to a few percent (4) and revealed, in combination with FTIR measurements, the protonation states of specific aspartate residues in various photocycle intermediates (5, 6). In the mutant D96N, the kinetics of proton uptake is affected (7-9). We recently showed that the low steady-state proton pumping act...

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate miceiles at pH 7.3. In the Asp-85 -Ala and Asp-85 --Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85 -* Ala and Asp-85 -* Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but dearly precedes the rise of M. The amplitude of the early (<0.2 ps) reversed photovoltage component in the Asp-85 -* Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212 -* Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 Ls, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82 -Gln mutant, no net transient proton release was observed, whereas, in the Arg-82 Ala mutant,, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85 --Glu, Arg-82-Ala, and Arg-82 --Gin mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85 -Ala, Asp-85 --Asn, and Asp-212 --Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.Site-directed mutagenesis has shown and Asp-212 to be essential for proton translocation by bacteriorhodopsin (bR) (1). The very low activity in mutants Asp-96 -+ Ala (D96A) and Asp-96 -* Asn (D96N) at pH 7 is due to a markedly slowed-down decay of the photocycle intermediate M and the associated charge movement (2-5). was concluded to be the internal proton donor for the reprotonation of the Schiff base (SB) leading to the decay of M (3, 4). Fourier-transform infrared spectroscopy has revealed the protonation states of Asp-85, Asp-96, Asp-115, and Asp-212 in the K, L, and M intermediates and provided clues to the time course of proton transfer (6, 7). The data suggest that Asp-85 and Asp-212 are deprotonated in the ground state and become protonated in the L -* M transition (6). A...

FTIR-difference spectroscopy in combination with site-directed mutagenesis has been used to investigate the role of water during the photocycle of bacteriorhodopsin. At least one water molecule is detected which undergoes an increase in H-bonding during the primary bR-->K phototransition. Bands due to water appear in the OH stretch region of the bR-->K FTIR-difference spectrum which downshift by approximately 12 cm-1 when the sample is hydrated with H2(18)O. In contrast to 2H2O, the H2(18)O-induced shift is not complete, even after 24 h of hydration. This indicates that even though water is still able to exchange protons with the outside medium, it is partially trapped in the interior of the protein. In the mutant Y57D, these bands are absent while a new set of bands appear at much lower frequencies which undergo H2(18)O-induced shifts. It is concluded that the water molecule we detect is located inside the bR active-site and may interact with Tyr-57. The change in its hydrogen-bonding strength is most likely due to the photoinduced all-trans-->13-cis isomerization of the retinal chromophore and the associated movement of the positively charged Schiff base during the bR-->K transition. In contrast, a second water molecule, whose infrared difference bands are not affected by the Y57D mutation, appears to undergo a decrease in hydrogen bonding during the K-->L and L-->M transitions.

We present a method that allows the detection of the surface charge density of bacteriorhodopsin (bR) at any selected protein surface site. The optical pH indicator fluorescein was covalently bound to the sulfhydryl groups of single cysteine residues, which were introduced at selected positions in bR by site-directed mutagenesis. On the extracellular side, the positions were in the BC loop (72) and in the DE loop (129-134). On the cytoplasmic side, one position in each loop was labeled: 35 (AB), 101 (CD), 160 (EF), and 231 (carboxy tail). The apparent pKs of fluorescein in these positions were determined for various salt concentrations. The local surface charge density was calculated from the dependence of the apparent pK of the dye on the ionic strength using the Gouy-Chapman equation. The surface charge density at pH 6.6 is more negative on the cytoplasmic side (averaged over all positions, -2.5 +/- 0.2 elementary charges per bR) than on the extracellular side (average, -1.8 +/- 0.2 elementary charges per bR) with little variation along the surface. Since the experiments were performed with electrically neutral CHAPS/DMPC micelles, these values represent the charge present on bR itself.(ABSTRACT TRUNCATED AT 250 WORDS)