Homologous to bacteriorhodopsin and even more to proteorhodopsin, xanthorhodopsin is a light-driven proton pump that, in addition to retinal, contains a noncovalently bound carotenoid with a function ...of a light-harvesting antenna. We determined the structure of this eubacterial membrane protein-carotenoid complex by X-ray diffraction, to 1.9-Å resolution. Although it contains 7 transmembrane helices like bacteriorhodopsin and archaerhodopsin, the structure of xanthorhodopsin is considerably different from the 2 archaeal proteins. The crystallographic model for this rhodopsin introduces structural motifs for proton transfer during the reaction cycle, particularly for proton release, that are dramatically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine-aspartate complex for regulating the pKa of the primary proton acceptor not present in archaeal pumps but apparently conserved in eubacterial pumps. In addition to aiding elucidation of a more general proton transfer mechanism for light-driven energy transducers, the structure defines also the geometry of the carotenoid and the retinal. The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as almost equal to45%, and the 46° angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
We produced the L intermediate of the photocycle in a bacteriorhodopsin crystal in photo-stationary state at 170 K with red laser illumination at 60% occupancy, and determined its structure to 1.62 A ...resolution. With this model, high-resolution structural information is available for the initial bacteriorhodopsin, as well as the first five states in the transport cycle. These states involve photo-isomerization of the retinal and its initial configurational changes, deprotonation of the retinal Schiff base and the coupled release of a proton to the extracellular membrane surface, and the switch event that allows reprotonation of the Schiff base from the cytoplasmic side. The six structural models describe the transformations of the retinal and its interaction with water 402, Asp85, and Asp212 in atomic detail, as well as the displacements of functional residues farther from the Schiff base. The changes provide rationales for how relaxation of the distorted retinal causes movements of water and protein atoms that result in vectorial proton transfers to and from the Schiff base.
Full text
Available for:
IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central ...issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here, we report on the X-ray diffraction structure of the L state, to 1.53–1.73 Å resolutions, from replicate data sets collected from six independent crystals. Unlike earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-
cis retinal in L is twisted at the C
13
=
C
14 and C
15
=
NZ double-bonds, and the Schiff base does not lose its connection to Wat402 and, therefore, to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L→M reaction is likely to occur, therefore,
via Wat402. It is evident from the structure of the L state that various conformational changes involving hydrogen-bonding residues and bound water molecules begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.
Full text
Available for:
IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The atomic structure of the light-driven ion pump bacteriorhodopsin and the surrounding lipid matrix was determined by X-ray diffraction of crystals grown in cubic lipid phase. In the extracellular ...region, an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base and the proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a π-bulge that causes a non-proline? kink. The bulge is stabilized by hydrogen-bonding of the main-chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The results indicate extensive involvement of bound water molecules in both the structure and the function of this seven-helical membrane protein. A bilayer of 18 tightly bound lipid chains forms an annulus around the protein in the crystal. Contacts between the trimers in the membrane plane are mediated almost exclusively by lipids.
Full text
Available for:
IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
An M intermediate of wild-type bacteriorhodopsin and an N intermediate of the V49A mutant were accumulated in photostationary states at pH 5.6 and 295 K, and their crystal structures determined to ...1.52
Å and 1.62
Å resolution, respectively. They appear to be M
1 and N′ in the sequence, M
1↔M
2↔M′
2↔N↔N′→O→BR, where M
1, M
2, and M′
2 contain an unprotonated retinal Schiff base before and after a reorientation switch and after proton release to the extracellular surface, while N and N′ contain a reprotonated Schiff base, before and after reprotonation of Asp96 from the cytoplasmic surface. In M
1, we detect a cluster of three hydrogen-bonded water molecules at Asp96, not present in the BR state. In M
2, whose structure we reported earlier, one of these water molecules intercalates between Asp96 and Thr46. In N′, the cluster is transformed into a single-file hydrogen-bonded chain of four water molecules that connects Asp96 to the Schiff base. We find a network of three water molecules near residue 219 in the crystal structure of the non-illuminated F219L mutant, where the residue replacement creates a cavity. This suggests that the hydration of the cytoplasmic region we observe in N′ might have occurred spontaneously, beginning at an existing water molecule as nucleus, in the cavities from residue rearrangements in the photocycle.
Full text
Available for:
IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Crystal structures of the Asp$^{96}$ to Asn mutant of the light-driven proton pump bacteriorhodopsin and its M photointermediate produced by illumination at ambient temperature have been determined ...to 1.8 and 2.0 angstroms resolution, respectively. The trapped photoproduct corresponds to the late M state in the transport cycle-that is, after proton transfer to Asp$^{85}$ and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. Its density map describes displacements of side chains near the retinal induced by its photoisomerization to 13-cis, 15-anti and an extensive rearrangement of the three-dimensional network of hydrogenbonded residues and bound water that accounts for the changed pK$_a$ values (where K$_a$ is the acid constant) of the Schiff base and Asp$^{85}$. The structural changes detected suggest the means for conserving energy at the active site and for ensuring the directionality of proton translocation.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
We illuminated bacteriorhodopsin crystals at 210
K to produce, in a photostationary state with 60% occupancy, the earliest M intermediate (M
1) of the photocycle. The crystal structure of this state ...was then determined from X-ray diffraction to 1.43
Å resolution. When the refined model is placed after the recently determined structure for the K intermediate but before the reported structures for two later M states, a sequence of structural changes becomes evident in which movements of protein atoms and bound water are coordinated with relaxation of the initially strained photoisomerized 13-
cis,15-
anti retinal. In the K state only retinal atoms are displaced, but in M
1 water 402 moves also, nearly 1
Å away from the unprotonated retinal Schiff base nitrogen. This breaks the hydrogen bond that bridges them, and initiates rearrangements of the hydrogen-bonded network of the extracellular region that develop more fully in the intermediates that follow. In the M
1 to M
2 transition, relaxation of the C
14–C
15 and C
15NZ torsion angles to near 180° reorients the retinylidene nitrogen atom from the extracellular to the cytoplasmic direction, water 402 becomes undetectable, and the side-chain of Arg82 is displaced strongly toward Glu194 and Glu204. Finally, in the M
2 to M
2′ transition, correlated with release of a proton to the extracellular surface, the retinal assumes a virtually fully relaxed bent shape, and the 13-methyl group thrusts against the indole ring of Trp182 which tilts in the cytoplasmic direction. Comparison of the structures of M
1 and M
2 reveals the principal switch in the photocycle: the change of the angle of the C
15NZ–CE plane breaks the connection of the unprotonated Schiff base to the extracellular side and establishes its connection to the cytoplasmic side.
Full text
Available for:
IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The K state, an early intermediate of the bacteriorhodopsin photocycle, contains the excess free energy used for light-driven proton transport. The energy gain must reside in or near the ...photoisomerized retinal, but in what form has long been an open question. We produced the K intermediate in bacteriorhodopsin crystals in a photostationary state at 100
K, with 40% yield, and determined its X-ray diffraction structure to 1.43
Å resolution. In independent refinements of data from four crystals, the changes are confined mainly to the photoisomerized retinal. The retinal is 13-
cis,15-
anti, as known from vibrational spectroscopy. The C
13C
14 bond is rotated nearly fully to
cis from the initial
trans configuration, but the C
14–C
15 and C
15NZ bonds are partially counter-rotated. This strained geometry keeps the direction of the Schiff base N–H bond vector roughly in the extracellular direction, but the angle of its hydrogen bond with water 402, that connects it to the anionic Asp85 and Asp212, is not optimal. Weakening of this hydrogen bond may account for many of the reported features of the infrared spectrum of K, and for its photoelectric signal, as well as the deprotonation of the Schiff base later in the cycle. Importantly, although 13-
cis, the retinal does not assume the expected bent shape of this configuration. Comparison of the calculated energy of the increased angle of C
12–C
13C
14, that allows this distortion, with the earlier reported calorimetric measurement of the enthalpy gain of the K state indicates that a significant part of the excess energy is conserved in the bond strain at C
13.
Full text
Available for:
IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The X-ray diffraction structure of the non-illuminated D96A bacteriorhodopsin mutant reveals structural changes as far away as 15 Å from residue 96, at the retinal, Trp-182, Ala-215, and waters 501, ...402, and 401. The Asp-to-Ala side-chain replacement breaks its hydrogen bond with Thr-46, and the resulting separation of the cytoplasmic ends of helices B and C is communicated to the retinal region through a chain of covalent and hydrogen bonds. The unexpected long-range consequences of the D96A mutation include breaking the hydrogen bond between O of Ala-215 and water 501 and the formation of a new hydrogen bond between water molecules 401 and 402 in the extracellular region. Because in the T46V mutant a new water molecule appears at Asp-96 and its hydrogen-bond to Ile-45 replaces Thr-46 as its link to helix B, the separation of helices B and C is smaller than that in D96A, and there are no atomic displacements elsewhere in the protein. Propagation of conformational changes along the chain between the retinal and Thr-46 had been observed earlier in the crystal structures of the D96N and E204Q mutants but in the trapped M state. Consistent with the perturbation of the retinal region in D96A, little change of the Thr-46 region occurs between the non-illuminated and M states of this mutant. It appears that a local perturbation can propagate along a track in both directions between the retinal and the Asp-96/Thr-46 pair, either from photoisomerization of the retinal in the wild-type protein in one case or from the D96A mutation in the other.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Proton pumps utilize a chemical or photochemical reaction to create pH and electrical gradients between the interior and the exterior of cells and organelles that energize ATP synthesis and the ...accumulation and extrusion of solutes and ions. G-protein coupled receptors bind agonists and assume signaling states that communicate with the coupled transducers. How these two kinds of proteins convert chemical potential to a proton transmembrane electrochemical potential or a signal are the great questions in structural membrane biology, and they may have a common answer. Bacteriorhodopsin, a particularly simple integral membrane protein, functions as a proton pump but has a heptahelical structure like membrane receptors. Crystallographic structures are now available for all of the intermediates of the bacteriorhodopsin transport cycle, and they describe the proton translocation mechanism, step by step and in atomic detail. The results show how local conformational changes propagate upon the gradual relaxation of the initially twisted photoisomerized retinal toward the two membrane surfaces. Such local−global conformational coupling between the ligand-binding site and the distant regions of the protein may be the shared mechanism of ion pumps and G-protein related receptors.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
PDF
