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Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein1

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Abstract

Replacement of aspartate 85 (D85) in bacteriorhodopsin (BR) by threonine but not be asparagine creates at pH<7 an anion-binding site in the molecular similar to that in chloride pump halorhodopsin. Binding of various anions to BR-D85T causes a blue shift of the absorption maximum by maximally 57 nm. Connected to this color change is a change in the absorption difference spectrum of the initial state and the longest living photo intermediate from a positive difference maximum at 460 nm in the absence of transported anions to one at 630 nm in their presence. Increasing anion concentration cause decreasing decay times of this intermediate. At physiological pH, BR-D85T but not BR-D85N transports chloride ions inward in green light, protons outward in blue or green light and protons inward in white light (directions refer to the intact cell). The proton movements are observable also in BR-D85N. Thus, creation of an anion-binding site in BR is responsible for chloride transport and introduction of anion-dependent spectroscopic properties at physiological pH. The different transport modes are explained with the help of the recently proposed IST model, which states that after light-induced isomerization of the retinal an ion transfer step and an accessibility change of the active site follow. The latter two steps occur independently. In order to complete the cyclic event, the accessibility change, ion transfer and isomerization state have to be reversed. The relative rates of accessibility changes and ion transfer steps define ultimately the vectoriality of ion transfers. All transport modes described here for the same molecule can satisfactorily be described in the framework of this general concept.

Introduction

The light-driven proton pump bacteriorhodopsin (BR) in the plasma membrane of Halobacteria is one of the best investigated transport proteins (for reviews, see Lanyi 1993, Ebrey 1993). The protein consists of seven α-helices forming a channel that is blocked in the middle by the covalently bound retinal. A structural model was suggested on the basis of an electron crystallographic analysis Henderson et al 1990, Grigorieff et al 1996 and a number of biophysical and biochemical investigations contributed to a principal understanding of the proton transport process in which aspartic acid residues 38 (D38), 85 (D85) and 96 (D96) play a central role for efficient proton translocation Riesle et al 1996, Mogi et al 1988, Braiman et al 1988, Gerwert et al 1989, Butt et al 1989, Holz et al 1989. The principal role of D85 in the extracellular half-channel is to accept a proton from the Schiff base in the first half of the catalytic cycle, while D96, located in the cytoplasmic half-channel, acts as an internal proton donor during reprotonation of the Schiff base in the second half. In spite of these established functions, proton translocation with wild-type vectoriality in blue light was demonstrated by BR mutants lacking both these aspartic acid residues indicating that they are not required in BR; neither for the proton translocation process per se nor for its vectoriality (Tittor et al., 1994). Surprisingly, the point mutated BR-D85T was shown to translocate chloride ions upon illumination with green light (Sasaki et al., 1995) in addition to its proton translocation capability in blue and white light (Tittor et al., 1994). The demonstration of different modes of ion translocation by the same molecule under different experimental conditions is one focus of the present work.

A second focus addresses the photochemical properties and the chloride dependence of BR-D85T. Neutralization of the D85 anion by protonation (acid BR or BR600 (Fischer & Oesterhelt, 1979), pKa ca 3) or mutation of D85 to asparagine or threonine induce a red shift of the absorption maximum (λmax) of BR from 570 nm to around 600 nm Marti et al 1991, Turner et al 1993, Tittor et al 1994, Sasaki et al 1995. Anion-dependent shifts of λmaxof the BR chromophore at low pH back to the original absorption maximum Fischer and Oesterhelt 1979, Drachev et al 1989, Varo and Lanyi 1989, resonance Raman experiments of the anion-bound form of BR (Diller et al., 1987) and alterations in the photo reaction dependent on anion concentration (Varo & Lanyi, 1989) suggested binding of chloride and other halides at a location close to the Schiff base and in the vicinity of D85. Mutants D85X, D85N/D212N and R82Q/D85N/D212N show red shifts and their absorption maxima similar to those observed with protonated D85. It was shown by resonance Raman and FTIR spectroscopy that the protonated Schiff base binds anions Marti et al 1992, Rath et al 1993.

The black lipid membrane technique (BLM) is a sensitive method to probe ion translocation by proteins and allows the identification of the transported ion by the use of ion-selective ionophores mediating stationary currents. Here, we demonstrate chloride and proton translocation in BR mutant D85T and the corresponding Cl-dependent alteration of the photochemical properties. Different modes of proton and chloride translocation activities are demonstrated under different experimental conditions such as the light quality and the ionic conditions. Molecular events leading to these different transport modes are discussed in the framework of the IST model introduced earlier (Haupts et al., 1997).

Section snippets

BR-D85T transports protons outward or inward and chloride inward

A convenient method to monitor electric events in light-driven ion pumps is the BLM technique (for a review, see Bamberg et al., 1993b). Purple membranes attach spontaneously in an oriented manner to a planar lipid membrane, separating two aqueous compartments of a BLM cell if added as suspension to one of the compartments and transient currents are detected with Ag/AgCl electrodes upon illumination. The occurrence of a light-induced current indicates electric coupling between the purple and

Conclusions

Introduction of threonine into the BR structure at position 85 introduces a variety of anion-dependent properties to the protein: anion-dependent shift of absorption maximum, anion-dependent exchanges of photocycle intermediates and Cl translocation. In addition, proton translocation in both directions by molecules with an initially deprotonated or protonated Schiff base is observed.

The results presented here can be rationalized in terms of the IST model of ion translocation by retinal

Strains

Construction of mutant strains D85N, D85T and isolation of mutated bacteriorhodopsins were as described Ferrando et al 1993, Tittor et al 1994.

Isolation of purple membranes

Isolation of purple membranes was according to the standard procedure (Oesterhelt & Stoeckenius, 1974). All three strains produce bacteriorhodopsin partly in the form of purple membranes, i.e. membrane fractions of buoyant density around 1.18g/cm3 which are isolated after water-induced lysis of the cells by sucrose density-gradient centrifugation. The

Acknowledgements

The skilful technical assistance of Bettina Brustmann is gratefully acknowledged. We thank Dr Martin Rüdiger and Professor Julian Schroeder for critical reading of the manuscript. The work was partially supported by SFB169 (to E.B.)

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