Bacteriorhodopsin Reconstituted From Two Individual Helices and the Complementary Five‐helix Fragment Is Photoactive

Kataoka, Mikio; Kahn, Theodore W.; Tsujiuchi, Yutaka; Engelman, Donald M.; Tokunaga, Fumio

doi:10.1111/j.1751-1097.1992.tb09710.x

Cited by 24 publications

(19 citation statements)

References 23 publications

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“…Bacteriorhodopsin helix assembly and activity is therefore largely governed by helical packing interactions alone, being remarkably insensitive to both structural perturbations of the inter-helical loops and to helical connectivity, consistent with previous observations (Kahn and Engelman 1992; Kataoka et al 1992; Marti 1998; Kim et al 2001). Currently, the principles of membrane protein folding are poorly understood relative to soluble proteins.…”

Section: Resultssupporting

confidence: 90%

An Empirical Test of Convergent Evolution in Rhodopsins

Mackin

Roy

Theobald

2013

Molecular Biology and Evolution

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Rhodopsins are photochemically reactive membrane proteins that covalently bind retinal chromophores. Type I rhodopsins are found in both prokaryotes and eukaryotic microbes, whereas type II rhodopsins function as photoactivated G-protein coupled receptors (GPCRs) in animal vision. Both rhodopsin families share the seven transmembrane α-helix GPCR fold and a Schiff base linkage from a conserved lysine to retinal in helix G. Nevertheless, rhodopsins are widely cited as a striking example of evolutionary convergence, largely because the two families lack detectable sequence similarity and differ in many structural and mechanistic details. Convergence entails that the shared rhodopsin fold is so especially suited to photosensitive function that proteins from separate origins were selected for this architecture twice. Here we show, however, that the rhodopsin fold is not required for photosensitive activity. We engineered functional bacteriorhodopsin variants with novel folds, including radical noncircular permutations of the α-helices, circular permutations of an eight-helix construct, and retinal linkages relocated to other helices. These results contradict a key prediction of convergence and thereby provide an experimental attack on one of the most intractable problems in molecular evolution: how to establish structural homology for proteins devoid of discernible sequence similarity.

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Section: Resultssupporting

confidence: 90%

An Empirical Test of Convergent Evolution in Rhodopsins

Mackin

Roy

Theobald

2013

Molecular Biology and Evolution

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“…Since helices B, C, and G are connected through a hydrogen bonding network (1), deprotonation of Schiff base (helix G) and protonation of Asp-85 (helix C) would perturb their interaction. In helix B, there are Tyr-57 and Tyr-64, which are expected to be hydrogen-bonded with residues in helix C. Therefore, it is plausible that helix B is affected by the environmental change generated near the Schiff base in the M intermediate earlier than helix F. bR reconstituted from two individual helices corresponding to A and B, and the remaining five-helix fragment, produces two different M photoproducts, also suggesting the importance of B helix at M formation (44).…”

Section: Discussionmentioning

confidence: 99%

Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction.

Kamikubo

Kataoka

Váró

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

115

146

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X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5°C. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The difference Fourier map indicates that the major change occurs near helix F. The observed diffraction changes between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin. Thus, we find that the protein conformations of the M and N intermediates of the photocycle are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated. The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surface and facilitate the proton transfer events that begin with the decay of the M state.The active site of an ion pump must communicate alternately with the two opposite sides of the membrane. Change of the protein conformation, linked to this switch, is therefore expected to be an essential step in the reaction cycle. In the light-driven proton pump bacteriorhodopsin (bR), the switch must occur after proton transfer toward the extracellular side but before proton transfer from the cytoplasmic side-i.e., while the proton binding site, the retinal Schiff base, is unprotonated. Thus, the switch reaction is expected to take place during the lifetime of the M intermediate in what was termed the Ml to M2 reaction (1-5). Structural changes are indeed revealed by neutron, x-ray, and electron diffraction when photostationary states are created where the M intermediate accumulates (6-9). We have shown that this structural change is closely related to the deprotonation of Schiff base; in the original structure (conformation E) the proton channel is open to the extracellular side, and when an M-type conformation is assumed (conformation C) it is open to the cytoplasmic side (10). Thus, the proton transport mechanism is elegantly explained by the alternating protein conformation model (11, 12). Light energy is utilized to cause the initial proton transfer that triggers the structural transition from conformation E to conformation C, and that results in the change of access of the retinal Schiff base as well as further changes in proton affinity (pK) of donor and acceptor groups so as to drive proton transfers at strategic locations in the protein.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" i...

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“…This assumption is supported by a number of studies that have shown that fragments of membrane proteins in which the loops between helices are disrupted or absent can retain the ability to self-assemble in a membrane (4). Experiments have shown that the bacteriorhodopsin molecule can be cut in several loops and reassembled (6,7), and related experiments on other proteins have demonstrated that coexpression of fragments cut in loop regions results in functional proteins inserted into membranes (see table in ref. 4).…”

Section: Resultsmentioning

confidence: 99%

Protein area occupancy at the center of the red blood cell membrane

Dupuy

Engelman²

2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

239

228

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In the Fluid Mosaic Model for biological membrane structure, proposed by Singer and Nicolson in 1972, the lipid bilayer is represented as a neutral two-dimensional solvent in which the proteins of the membrane are dispersed and distributed randomly. The model portrays the membrane as dominated by a membrane lipid bilayer, directly exposed to the aqueous environment, and only occasionally interrupted by transmembrane proteins. This view is reproduced in virtually every textbook in biochemistry and cell biology, yet some critical features have yet to be closely examined, including the key parameter of the relative occupancy of protein and lipid at the center of a natural membrane. Here we show that the area occupied by protein and lipid at the center of the human red blood cell (RBC) plasma membrane is at least Ϸ23% protein and less than Ϸ77% lipid. This measurement is in close agreement with previous estimates for the RBC plasma membrane and the recently published measurements for the synaptic vesicle. Given that transmembrane proteins are surrounded by phospholipids that are perturbed by their presence, the occupancy by protein of more than Ϸ20% of the RBC plasma membrane and the synaptic vesicle plasma membrane implies that natural membrane bilayers may be more rigid and less fluid than has been thought for the past several decades, and that studies of pure lipid bilayers do not fully reveal the properties of lipids in membranes. Thus, it appears to be the case that membranes may be more mosaic than fluid, with little unperturbed phospholipid bilayer.fluid mosaic model ͉ membrane lipid bilayer T he protein-to-lipid ratio has often been measured, but such measures do not address the question we raise here, because the proteins usually extend well beyond the bilayer and often occupy surface area-the finding from the recent modeling of the synaptic vesicle is that there is little lipid exposed, being covered by extrabilayer protein domains (1). There is great variability in the measured protein-to-lipid ratios, from myelin, with Ϸ20% protein, to the inner mitochondrial membrane at 76% protein; the plasma membranes of most animal cells are Ϸ50% protein. Nonetheless, with the exception of the purple membrane from Halobacterium salinarium, there is little accurate information concerning the area occupancy of protein and lipid at the center of natural membranes (2, 3).

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Bacteriorhodopsin Reconstituted From Two Individual Helices and the Complementary Five‐helix Fragment Is Photoactive

Cited by 24 publications

References 23 publications

An Empirical Test of Convergent Evolution in Rhodopsins

An Empirical Test of Convergent Evolution in Rhodopsins

Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction.

Protein area occupancy at the center of the red blood cell membrane

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