BR - Bacteriorhodopsin

Bacteriorhodopsin

I. Introduction

The retinal protein bacteriorhodopsin is the major photosynthetic protein of the archaeon Halobacterium salinarum. It converts the energy of "green" light (500-650 nm, max 568 nm) into an electrochemical proton gradient, which in turn is used for ATP production by ATP synthase. It functions as a light-driven proton pump, transporting protons out of the cell, and exemplifies vectorial catalysis.

Bacteriorhodopsin is the focus of much interest and has become a paradigm for membrane proteins in general and transporters in particular. Its structure and function have been analyzed in great detail using a multitude of different experimental techniques and has become the best-understood example of vectorial catalysis.

Bacteriorhodopsin is also involved in phototaxis (Bibikov, 1993, Bibikov, 1991, Grishanin, 1996) via the generation of membrane potential changes (ΔΔΨ) across the membrane which are sensed by transducer mcpT (Koch, 2005).

The reversible light-triggered color-change has allowed to develop biotechnological applications of bacteriorhodopsin, e.g. its application in optical information recording.

The further description is based on the review article by Haupts, Tittor, and Oesterhelt (1999), Oesterhelt (1998) and Oesterhelt(1999).

II. Structure and function of bacteriorhodopsin

Fig. 1: the 3D structure of bacteriorhodopsin (cross section of the structural model PDB:1BRR. Selected residues important for proton transfer steps are marked. The probable path of protons is indicated by arrows. Zoom Image
Fig. 1: the 3D structure of bacteriorhodopsin (cross section of the structural model PDB:1BRR. Selected residues important for proton transfer steps are marked. The probable path of protons is indicated by arrows. [less]

Bacteriorhodopsin - as all retinal proteins from Halobacterium - folds into a seven-transmebrane helix topology with short interconnecting loops. The helices (named A-G) are arranged in an arc-like structure and tightly surround a retinal molecule that is covalently bound via a Schiff base to a conserved lysine (Lys-216) on helix G. The cross-section of BR with residues important for proton transfer and the probable path of the proton is shown in Fig.1. The 3D structure is accessible through the PDB database, entry 1BRR.

Fig. 2: the sequence of bacteriorhodopsin written to show the seven transmembrane helices and the extracellular and intracellular loops. The extracellular surface is at the bottom. Zoom Image
Fig. 2: the sequence of bacteriorhodopsin written to show the seven transmembrane helices and the extracellular and intracellular loops. The extracellular surface is at the bottom. [less]

Fig. 2 shows a model of the sequence.

Retinal separates a cytoplasmic from an extracellular half channel that is lined by amino acids crucial for efficient proton transport by BR (especially Asp-96 in the cytoplasmic and Asp-85 in the extracellular half channel). The Schiff base between retinal and Lys-216 is located at the center of this channel. To allow vectorial proton transport, de- and reprotonation of the Schiff base must occur from different sides of the membrane. Thus, the accessibility of the Schiff base for Asp-96 and Asp-85 must be switched during the catalytic cycle. The geometry of the retinal, the protonation state of the Schiff base, and its precise electrostatic interaction with the surrounding charges (Asp-85, Asp-212, Arg-82) and dipoles tune the absorption maximum to fit its biological function.

III. The purple membrane

The surface of Halobacterium salinarum contains membrane patches called the purple membrane. The protein:lipid ratio is 75:25. The only protein in the purple membrane is bacteriorhodopsin which forms a hexagonal 2-dimensional crystal consisting of bacteriorhodopsin trimers.

The purple membrane can be easily isolated and permits mass production of bacteriorhodopsin as is required for biotechnological applications.

IV. The catalytic cycle of bacteriorhodopsin

Fig. 3: the photocycle of bacteriorhodopsin Zoom Image
Fig. 3: the photocycle of bacteriorhodopsin

Absorption of a photon by bacteriorhodopsin initiates a catalytic cycle that leads to transport of a proton out of the cell. Several intermediates in the photocycle have been identified by spectroscopic techniques (Fig.3). By application of a multitude of biophysical techniques, the exact nature of the changes in each step of the cycle has been determined and has been related to transport function.

The cycle can be formally described in terms of six steps of isomerization (I), ion transport (T), and accessibility change (switch S). Retinal first photo-isomerizes from an all-trans to a 13-cis configuration followed by a proton transfer from the Schiff base to the proton acceptor Asp-85. To allow vectoriality, reprotonation of the Schiff base from Asp-85 must be excluded. Thus, its accessibility is switched from extracellular to intracellular. The Schiff base is then reprotonated from Asp-96 in the cytoplasmic channel. After reprotonation of Asp-96 from the cytoplasmic surface, retinal reisomerizes thermally and the accessibility of the Schiff base switches back to extracellular to reestablish the initial state. These steps represent the minimal number of steps needed to account for vectorial catalysis in wild-type bacteriorhodopsin.

V. The catalytic cycle step by step

Dynamic structural changes occuring in chromophore and protein during the light-induced reaction cycle can be detected either directly by time-resolved spectroscopic techniques (ultrafast laser spectroscopy, flash photolysis, ESR spectroscopy, FTIR spectroscopy) or by trapping intermediate states, determining their structures by static methods (NMR spectroscopy, electron microscopy, neutron scattering) and comparing it with the ground state.

  • primary reaction: the photoisomerization of retinal from all- trans to 13- cis In a stereoselective process, all- trans retinal is photoisomerized to 13- cis retinal. This process has been time-resolved to few femtoseconds. Within 500 fs, all- trans retinal isomerizes to 13- cis retinal, resulting in J600 which is converted to K590 within another 5 ps.
  • from the K590 to the L550 intermediate The K590 intermediate is transformed to the L550 intermediate within 2 µs. The hydrogen bonding interaction in the extracellular channel between the protonated Schiff base and Asp-85, which involves a water molecule, is strengthened.
  • first proton translocation step: from L550 to M410(EC) The M state is reached from the L state within several microseconds. This transition involves transfer of a proton from the Schiff base to Asp-85 in the extracellular half-channel.
  • first accessibility switch reaction from extracellular to cytoplasmic: M410(EC) to M410(CP) To allow vectorial proton transport, de- and reprotonation of the Schiff base must occur from different sides of the membrane. This accessibility switch occurs at the level of the M intermediate: M410(EC) to M410(CP). Thus, the originally described "M" intermediate is in fact split into two or more different intermediates all having yellow color.
  • second proton transfer step: from M410(EC) to N560 Reprotonation of the Schiff base from Asp-96 in the cytoplasmic half-channel occurs during transformation from the M410(EC) to the N560 intermediate within milliseconds. Reprotonation of Asp-96 by a proton from the cytoplasm also occurs during the lifetime of the N560 intermediate.
    It should be noted that Asp-96 functions as a proton storage for reprotonation of the Schiff base. Therefore, the proton does not originate directly from the cytoplasm. This detail solves the puzzling phenomenon that the transport rate of this proton transporter is not pH-dependent (within limits).
  • thermoisomeration of retinal from 13- cis to all- trans : N560 to O640 The transition of the N560 to the O640 intermediate is the thermal 13- cis to all- trans isomerization of retinal in the environment of protonated Asp-96 and protonated Asp-85.
  • second accessibility switch reaction from cytoplasmic to extracellular: O640 to BR Deprotonation of Asp-85 completes the catalytic cycle. Switching the accessibility of the Schiff base back from extracellular to intracellular occurs within ca 5 ms and results in restoration of the initial state.

VI. Site-directed mutagenesis of bacteriorhodopsin

An important tool for these studies, and also for spectroscopic investigations on structure and dynamics, is the possibility to produce specifically modified proteins by site directed mutagenesis and homologous overexpression. By this method the role of individual amino acid residues for the transport mechanism can be investigated, or reporter molecules can be introduced at certain positions. Several mutants interfere with the photocycle and and proton transport and may permit to trap intermediates of the catalytic cycle. The direct involvement of Asp-85 and Asp-96 in proton transfer has been demonstrated by analysis of mutants.

VII. Bacteriorhodopsin as material for optical information recording

Biotechnological applications on the basis of the colour change between purple and yellow (long living intermediate M) is the basis for using of bacteriorhodopsin for optical information recording. The technique has advanced such that it could be used as a safety feature on chipcards.

For further details see the page on bacteriorhodopsin on the web site of Prof. N. Hampp, University of Marburg (external link)

VIII. Single molecule technology applied to the analysis of bacteriorhodopsin

Atomic force spectroscopy has been used to handle single molecules of bacteriorhodopsin in the purple membrane. This has permitted to measure the force required to pull individual helices out of the membrane.

One of the molecular prerequisites for these experiments was the introduction of Cys residues into bacteriorhodopsin by site-directed mutagenesis.

For further details see Prof. H.E. Gaub, LMU Munich (external link)

IX. More detailed analysis in cooperations with other groups

IX. More detailed analysis in cooperations with other groups

In cooperation with many external scientists, the very detailed analysis of bacteriorhodopsin has been described in a plethora of publications. The description of these details is beyond the scope of this web site.

Cooperation partners are:

E. Bamberg

N.A. Dencher

H.E. Gaub

K. Gerwert

M. Gutman

N. Hampp

J. Heberle

R. Henderson

D.J. Müller

P. Schmieder

H.-J. Steinhoff

G. Zaccai

W. Zinth

References:

Review article

  • U. Haupts, J. Tittor, D. Oesterhelt:
    Closing in on Bacteriorhodopsin: Progress in Understanding the Molecule.
    Ann. Rev. Biophys. Biomol. Struct. 28, 367-399 (1999)
  • D. Oesterhelt:
    The Structure and Mechanism of the Family of Retinal Proteins from Halophilic Archaea Curr. Op.
    Struct. Biol. 8, 489-500 (1998)
  • D. Oesterhelt:
    Photosynthese der zweiten Art: Die halophilen Archaea.
    In: Photosynthese (D. Häder, ed.) Georg Thieme Verlag, Stuttgart (1999) pp. 146-161.

3D structure

photocycle

NMR spectroscopy

pico- and femtosecond spectroscopy

  • P. Hamm, M. Zurek, T. Röschinger, H. Patzelt, D. Oesterhelt and W. Zinth:
    Femtosecond Spectroscopy on the Photoisomerisation of the Protonated Schiff Base of all-trans Retinal.
    Chem. Phys. Lett. 263, 613-621 (1996)
  • J. Dobler, W. Zinth, W. Kaiser, D. Oesterhelt:
    Excited-State Reaction Dynamics of Bacteriorhodopsin Studied by Femtosecond Spectroscopy.
    Chem. Phys. Lett. 144, 215-220 (1988).
  • H.-J. Polland, M.A. Franz, W. Kaiser, D. Oesterhelt:
    Energy Transfer from Retinal to Amino Acids. - A Time-Resolved Study of the Ultraviolet Emission of Bacteriorhodospin.
    Biochim. Biophys. Acta 851, 407-415 (1986).
  • H.-J. Polland, M.A. Franz, W. Zinth, W. Kaiser, E. Kölling, D. Oesterhelt:
    Optical Picosecond Studies of Bacteriorhodopsin Containing a Sterically Fixed Retinal.
    Biochim. Biophys. Acta 767, 635-639 (1984).

site-directed mutagenesis

biotechnological application

nanotechnology

involvement in phototaxis

further references and classical papers

 
loading content
Go to Editor View