Purple membrane structure

Olle Edholm, Oliver Berger, and Fritz Jahnig. Structure and fluctuations of bacteriorhodopsin in the purple membrane. J. Mol. Biol., 250 94 111, 1995. 

Indeed, hydrophilic N- or C-terminal ends and loop domains of these membrane proteins exposed to aqueous phases are able to undergo rapid or intermediate motional fluctuations, respectively, as shown in the 3D pictures of transmembrane (TM) moieties of bacteriorhodopsin (bR) as a typical membrane protein in the purple membrane (PM) of Halobacterium salinarum.176 178 Structural information about protein surfaces, including the interhelical loops and N- and C-terminal ends, is completely missing from X-ray data. It is also conceivable that such pictures should be further modified, when membrane proteins in biologically active states are not always present as oligomers such as dimer or trimer as in 2D or 3D crystals but as monomers in lipid bilayers. 

Figure 5. Chemical structures of main lipids of purple membranes from Halobacterium salinarium S9 phosphatidylglycerophosphate (PGP), phosphatidylglycerol (PG) and glycolipid sulfate (GLS).

The following analysis and discussion of protein structure is based almost exclusively on the results of three-dimensional X-ray crystallography of globular proteins. In addition, one structure is included that was determined by electron diffraction (purple membrane protein), and occasional reference is made to particularly relevant results from other experimental techniques or from theoretical calculations. Even with this deliberately restricted viewpoint the total amount of information involved is immense. Millions of independent parameters have been determined by protein crystallography, and the relationships among almost any subset of them are of potential interest. A major aim of the present study is to provide a guide map for use in exploring this forest of information. 

In HREM images of inorganic crystals, phase information of structure factors is preserved. However, because of the effects of the contrast transfer function (CTF), the quality of the amplitudes is not very high and the resolution is relatively low. Electron diffraction is not affected by the CTF and extends to much higher resolution (often better than lA), but on the other hand no phase information is available. Thus, the best way of determining structures by electron crystallography is to combine HREM images with electron diffraction data. This was applied by Unwin and Henderson (1975) to determine and then compensate for the CTF in the study of the purple membrane. 

Vitamin A is necessary for growth and reproduction, resistance to infection, maintenance and differentiation of epithelial tissues, stability and integrity of membrane structures, and the process of vision. In terms of the last function, vitamin A is a component of rhodopsin or visual purple, a photosensitive pigment in the eye that is needed for vision in dim light. An early mild clinical symptom of vitamin A deficiency is night blindness a severe deficiency of this fat-soluble vitamin results in xerophthalmia, an eye condition leading to blindness. 

Ordered arrangements of proteins in membranes in vivo With the exception of the purple membrane produced by Halobacterium halobium, the order discussed in this section does not involve an exact regular geometrical structure. However, membrane bound enzymes and structure proteins associated with membranes are arranged in ways which are clearly defined and which involve definite and regular interactions between different kinds of molecule. Only thus can they carry out... 

DzO have shown how the water molecules are distributed in the structure in different conditions (Zaccai, 1987 Papadopoulos et al., 1990). In its halophilic physiological environment, the membrane has a multimolar KC1 solution on its cytoplasmic side and a multimolar NaCl solution on the outside of the cell. Most structure and function experiments on purple membranes, however, have been done in low salt concentration conditions. Neutron diffraction experiments have been attempted in high concentrations of KC1 and NaCl, but results are not yet available (F. Samatey and G. Zaccai, unpublished data). 

Many of the early genetic studies were done on H. halobium and its related strains. The popularity of these strains stemmed from the fact that several interesting spontaneous mutations could be readily detected. Among the most characterized mutations are those that affect the production of the protein part of the purple membrane, the heavily studied light-driven proton pump bacteriorhodopsin. Spontaneous mutations occur at a frequency of lO 4. Analysis of these mutations showed that in almost every case a foreign DNA sequence was introduced into the bacterioopsin (bop) structural gene or into sequences surrounding it. 

Figure 10-4. Controlled extraction of an individual BR from native purple membrane. (A) Typical high-resolution AFM topograph of the cytoplasmic surface of a wild-type purple membrane. (B) The stylus and protein surface were separated at a velocity of 40 nm/s while the force spectrum was recorded (512 or 4096 pixels). The interaction between tip and surface, which is expressed in the marked discontinuous changes in the force, indicates a molecular bridge between tip and sample. This bridge reaches far out to distances up to 75 nm, which corresponds to the length of one totally unfolded protein. (C) After the adhesive force peaks were recorded, a topograph of the same surface was taken to show structural changes.23...

Belrhali H, Nollert P, Royant A, Menzel C, Rosenbusch JP, Landau EM, Pebay-Peyroula E (1999) Protein, lipid, and water organization in bacteriorhodopsin crystals A molecular view of the purple membrane at 1. 9 A. Structure Fold. Des. 7 909-917... 

Belrhali, H., Nollert, P., Royant, A., Menzel, C., Rosenbusch, J. P., Landau, E. M., and Pebay-Peyroula, E. (1999). Protein, lipid and water organization in bacteriorhodopsi n crystals a molecular view of the purple membrane at 1.9 A resolution. Structure Fold. Des. 7, 909-917. 

An additional basic difference between rhodopsin and bacter-iorhodopsin is associated with the structure of the pigment in the membrane. In contrast to the well-established rotational and translational mobility of the rhodopsin molecule in the viscous disk membrane (31-35), X-ray diffraction methods have shown that bacteriorhodopsin in the purple membrane is organized as a rigid two-dimensional hexagonal lattice with a 63 A unit cell (36-38). ... 

On the basis of novel electron diffraction methods, Unwin and Henderson have shown that each protein molecule consists of seven ot-hexical rods, about 40 A long and 10 A apart, all perpendicular to the plane of the membrane (39,40). The lattice structure results in clustering of the BR molecules as trimers around one of the threefold axes. Analysis of CD spectra in terms of exciton interactions between the chromophores (41,42), as well as linear dichroism measurements (43-45), have yielded values of 20-24° for the tilt angle (out of the membrane plane) of the retinal transition moment, which closely coincides with the long axis of the molecule. The absence of any rotational mobility of bacteriorhodopsin in the purple membrane (on a time scale of 60 min) was also confirmed by linear dichroism measurements (45). 

Bacteriorhodopsin, is a retinal-containing protein in the purple membrane of a halophilic, (salt-loving) archaebacterium, Halobacterium halobium, which pumps protons out of the cell on activation by light.The three-dimensional structure of bacteriorhodopsin resembles that of rhodopsin in the eye. 

Photoreceptor Pigments. There have been several reviews on the structures, photochemistry, and functioning of the retinal-protein photoreceptor pigments involved in the processes of visionand in the purple membrane of Halobacteria (bacteriorhodopsin). ° ° In addition to the papers quoted earlier on the spectroscopy of these pigments, many other reports have appeareddealing with rhodopsin and intermediates in its photocycle, especially photochemistry, chromophore-protein conformation and binding, and reaction kinetics. Similar studies on bacteriorhodopsin have also been described." "-"" ... 

Cells lack the intracellular proliferation of unit-membrane structures that bear the majority of pigments in purple bacteria. 

The protein (26kDa) is a bundle of seven helices, A through G, with three helices normal to the membrane plane and the rest inclined at small angles to the normal. The retinal is covalently bound through a protonated Schiff base to Lys-216 at the middle of helix G, and it lies nearly parallel to the membrane in the space surrounded by the seven helices. The protein forms in vivo a homotrimer, and the trimers are assembled into an extended hexagonal lattice (P3 symmetry), the purple membrane. Protein-protein contact in these patches is through a continuous boundary layer of lipids no more than one lipid wide, at the monomer periphery. The interhelical loops as well as the N- and C-termini are short, with the exception of the connection of helices B and C through a structured fS-turn on the extracellular side, and the E-F interhelical loop with a twist, on the cytoplasmic side. 

A complex multi-chromophoric system comprises the purple membrane patches from Halobacterium salinarium. These patches are composed of about 3000 bacter-iorhodopsin proteins. The hyperpolarizability of solubilized monomeric bacterio-rhodopsin was measured by HRS and found to be 2100 x 10 esu at 1064 nm. This high value is due to the presence of a chromophore in the protein, the proto-nated Schiff base of retinal. A purple membrane patch can be treated as a two-dimensional crystal of bacteriorhodopsin proteins, and its structure is known in considerable detail. The analysis of the purple membrane tensor was performed by adding the hyperpolarizabilities of the individual proteins in the purple membrane. From (depolarized) HRS measurements on purple membrane suspensions, the structure of the purple membrane patches, and an average membrane size measured by atomic force microscopy, a fi value of 2200 x 10 esu was calculated for bacteriorhodopsin [22]. The organization of the dipolar protonated Schiff base chro-mophores in the membranes was found to be predominantly octopolar. 

The purple membrane of Halobacterium halobium was discovered in the late 1960 s when Stoeckenius and co-workers described sheet-like structures which could... 

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