Bacteriorhodopsin three-dimensional structure

Halorhodopsiti. In addition to bacteriorhodopsin there are three other retinal-containing proteins in membranes of halobacteria. From mutant strains lacking bacteriorhodopsin the second protein, halorhodopsin, has been isolated. It acts as a light-driven chloride ion pump, transporting Cl from outside to inside. Potassium ions follow, and the pump provides a means for these bacteria to accumulate KC1 to balance the high external osmotic pressure of the environment in which they live.578 The amino acid sequences of halorhodopsins from several species are very similar to those of bacteriorhodopsin as is the three-dimensional structure.589 However, the important proton-carrying residues D85 and D96 of bacteriorhodopsin are replaced by threonine and alanine, respectively, in halorhodopsin.590 Halorhodopsin (hR)... 

A three-dimensional structure also has been elucidated for bacteriorhodopsin, an integral membrane protein of the halophilic (salt-loving) bacterium Halobacterium halobium. This protein has been studied intensively because of its remarkable activity as a light-driven proton pump (see chapter 14). It forms well-ordered arrays in two-dimensional sheets that can be studied by electron diffraction. Measurements of the diffraction patterns show clearly that bacteriorhodopsin has seven transmembrane helices (fig. 17.12). 

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. 

The exact location of the Schiff base and the Asp-96 carboxyl is yet to be found since the three-dimensional structure of bacteriorhodopsin is not known to atomic resolution. Electron microscopy of two-dimensional bacteriorhodopsin crystals indicates that the Schiff base is localized in the middle of the protein molecule, while Asp-96 is somewhere between the Schiff base and the cytoplasmic surface of the membrane [21]. It has also been shown that the protein regions separating the Schiff base from the outer and cytoplasmic membrane surfaces differ strongly in hydrophobicity, which is low in the former, and high in the latter case. Between the outer membrane surface and the Schiff base, there are four charged amino acids and no valine, leucine and isoleucine. At the same time, between the Schiff base and the cytoplasm, five leucines, valine and only one charged amino acid (Asp-96) seem to be localized [21]. Thus the dielectric... 

No direct three-dimensional structure determination of any GPCR has been carried out yet. However, the structure of the membrane-embedded helices of bacteriorhodopsin has been solved by electron crystallography [131]. Experimental data from deletion mutations, antibody targeting, and proteolytic digestion experiments provide evidence that the overall features of GPCRs and... 

M FIGURE 5-13 Structural model of bacteriorhodopsin, a multipass transmembrane protein that functions as a photoreceptor in certain bacteria. The seven hydrophobic a helices in bacteriorhodopsin traverse the lipid bilayer. A retinal molecule (red) covalently attached to one helix absorbs light. The large class of G protein-coupled receptors in eukaryotic cells also has seven membrane-spanning a helices their three-dimensional structure is similar to that of bacteriorhodopsin. [After H. Luecke et al., 1999, J. Mol. Biol. 291 899.]... 

Notwithstanding this complexity, the need for three-dimensional, structural information at the atomic level of resolution is central and indispensable to biomembrane science. X-ray, and to a lesser extent neutron-diffraction, as the most important sources for such information have, therefore, been widely used in this field (for reviews, see Refs. 1-4). The success of this approach, however, has generally been less spectacular than for instance in the cases of protein or nucleic acid structure. The reasons for this lie in the very nature of biological membranes with few, notable exceptions (such as the purple membrane of halobacterium halobium, which can be viewed essentially as a two-dimensional crystal of bacteriorhodopsin with only little lipid. Refs. 5, 6,25) biological membranes are characterized by highly complex and variable molecular compositions, and by the structural dynamics, fluidity , which is in many cases essential for enzymatic, or other, functions of membranes. As a reflection of this most natural membranes do not crystallize, and a full, three-dimensional atomic structure analysis seems out of reach. 

Fig. 3. Three-dimensional structure of bacteriorhodopsin from 1.5 A resolution as determined by X-ray diffraction25 (B) and a top view of hexagonal packing (A).45a Reproduced with permission from Academic Press Ltd.

Site-directed mutagenesis as applied to bacteriorhodopsin was a difficult and labor-intensive procedure that used E. coli as the expression system (79, 80). Recently, Needleman s group successfully developed a new expression system that uses a bacteriorhodopsin-deficient mutant strain of H. halobium (43, 44). Preliminary results were quite encouraging. Unlike the mutants expressed in E. coli, the new method produces mutant bacteriorhodopsins with properties that differ from the protein expressed by E. coli. Presumably this difference occurs because correct folding into three-dimensional structures is more likely in the natural host than in its surrogate. Denaturation of the mutant proteins is further avoided because reconstitution is unnecessary. Our preliminary results show that the fast photoelectric signal can be drastically altered by a judiciously chosen point mutation. 

It has been hard to determine the three-dimensional structure of rhodopsin or other receptors of this family. However, their relationship to bacteriorhodopsin, whose structure was obtained in 1975 by electron crystallography and more recently by X-ray crystallography at... 

Figure 4.4 (a) Schematic representation of the three-dimensional structure of bacteriorhodopsin (bR). (b) Photoisomerization of all-trans to 13-cis retinal in bR. 

Cartoon representation of the three-dimensional structures of three different photoresponsive proteins. Left GFP with its HBDI anionic chromophore. Center the bovine visual pigment rhodopsin together with the cationic PSB11 chromophore. Right bacteriorhodopsin and its PSBAT chromophore... 

Figure 12.3 Two-dimensional crystals of the protein bacteriorhodopsin were used to pioneer three-dimensional high-resolution structure determination from electron micrographs. An electron density map to 7 A resolution (a) was obtained and interpreted in terms of seven transmembrane helices (b).

The crystallization of the photosynthetic reaction centre was crucial for the structure determination. One has to keep in mind that the photosynthetic reaction centre is a complex of membrane proteins, and it was considered to be impossible to crystallize membrane proteins at that time. The reaction centre was therefore the first membrane protein or complex of membrane protein whose structure could be determined. I had started the crystallization of membrane proteins in 1978 when working as a post doc in Dieter Oesterhelt s lab in Wurzburg. My attempts were caused by an accidental observation, which I had made with bacteriorhodopsin and which led me to try to crystallize membrane proteins and to develop strategies to achieve this. The results were two papers in the Proceedings of the National Aeademy of Sciences of the U.S.A. with D. Oesterhelt in 1980 (77, 338-342 77, 1283-1285). One described the formation and analysis of a new two-dimensional crystal form of bacteriorhodopsin and the other was about the first true three-dimensional crystallization of any membrane protein, namely bacteriorhodopsin. 

The sequence similarities between bacteriorhodopsins and halorhodopsins from various species [17,43—45] aigue for extensive structural similarities between the two kinds of proteins. It seems likely that the tertiary structure of the halorhodopsins is a generalized bacteriorhodopsin-like arrangement, but two- or three-dimensional crystalline halorhodopsin has not been produced as yet. A crude model for halorhodopsin, based on... 

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