Crystallographic structures of the M and N intermediates of bacteriorhodopsin: assembly of a hydrogen-bonded chain of water molecules between Asp-96 and the retinal Schiff base

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 1.52A and 1.62A 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.     

Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin

In order to understand how isomerization of the retinal drives unidirectional transmembrane ion transport in bacteriorhodopsin, we determined the atomic structures of the BR state and M photointermediate of the E204Q mutant, to 1.7 and 1.8 A resolution, respectively. Comparison of this M, in which proton release to the extracellular surface is blocked, with the previously determined M in the D96N mutant indicates that the changes in the extracellular region are initiated by changes in the electrostatic interactions of the retinal Schiff base with Asp85 and Asp212, but those on the cytoplasmic side originate from steric conflict of the 13-methyl retinal group with Trp182 and distortion of the pi-bulge of helix G. The structural changes suggest that protonation of Asp85 initiates a cascade of atomic displacements in the extracellular region that cause release of a proton to the surface. The progressive relaxation of the strained 13-cis retinal chain with deprotonated Schiff base, in turn, initiates atomic displacements in the cytoplasmic region that cause the intercalation of a hydrogen-bonded water molecule between Thr46 and Asp96. This accounts for the lowering of the pK(a) of Asp96, which then reprotonates the Schiff base via a newly formed chain of water molecules that is extending toward the Schiff base.     

Conformational change of helix G in the bacteriorhodopsin photocycle: investigation with heavy atom labeling and x-ray diffraction.

According to the current structural model of bacteriorhodopsin, Ile222 is located at the cytoplasmic end of helix G. We labeled the single cysteine of the site-directed mutant Ile222 --> Cys with p-chloromercuribenzoic acid and determined the position of the labeled mercury by x-ray diffraction in the unphotolyzed state, and in the MN photointermediate accumulated in the presence of guanidine hydrochloride at pH 9.5. According to the difference Fourier maps between the MN intermediate and the unphotolyzed state, the structural change in the MN intermediate was not affected by mercury labeling. The difference Fourier map between the labeled and the unlabeled I222C gave the position of the mercury label. This information was obtained for both the unphotolyzed state and the MN intermediate. We found that the position of the mercury at residue 222 is shifted by 2.1 +/- 0.8 A in the MN intermediate. This agrees with earlier results that suggested a structural change in the G helix. The movement of the mercury label is so large that it must originate from a cooperative conformational change in the helix G at its cytoplasmic end, rather than from displacement of residue 222. Because Ile222 is located at the same level on the z coordinate as Asp96, the structural change in the G helix could have the functional role of perturbing the environment and therefore the pKa of this functionally important aspartate.     

Binding of calcium ions to bacteriorhodopsin.

Adding Ca2+ or other cations to deionized bacteriorhodopsin causes a blue to purple color shift, a result of deprotonation of Asp85. It has been proposed by different groups that the protonation state of Asp85 responds to the binding of Ca2+ either 1) directly at a specific site in the protein or 2) indirectly through the rise of the surface pH. We tested the idea of specific binding of Ca2+ and found that the surface pH, as determined from the ionization state of eosin covalently linked to engineered cysteine residues, rises about equally at both extracellular and cytoplasmic surfaces when only one Ca2+ is added. This precludes binding to a specific site and suggests that rather than decreasing the pKa of Asp85 by direct interaction, Ca2+ increases the surface pH by binding to anionic lipid groups. As Ca2+ is added the surface pH rises, but deprotonation of Asp85 occurs only when the surface pH approaches its pKa. The nonlinear relationship between Ca2+ binding and deprotonation of Asp85 from this effect is different in the wild-type protein and in various mutants and explains the observed complex and varied spectral titration curves.     

Reconstitution of gloeobacter rhodopsin with echinenone: role of the 4-keto group

In previous work, we reconstituted salinixanthin, the C(40)-carotenoid acyl glycoside that serves as a light-harvesting antenna to the light-driven proton pump xanthorhodopsin, into a different protein, gloeobacter rhodopsin expressed in Escherichia coli, and demonstrated that it transfers energy to the retinal chromophore [Imasheva, E. S., et al. (2009) Biochemistry 48, 10948]. The key to binding of salinixanthin was the accommodation of its ring near the retinal β-ionone ring. Here we examine two questions. Do any of the native Gloeobacter carotenoids bind to gloeobacter rhodopsin, and does the 4-keto group of the ring play a role in binding? There is no salinixanthin in Gloeobacter violaceous, but a simpler carotenoid, echinenone, also with a 4-keto group but lacking the acyl glycoside, is present in addition to β-carotene and oscillol. We show that β-carotene does not bind to gloeobacter rhodopsin, but its 4-keto derivative, echinenone, does and functions as a light-harvesting antenna. This indicates that the 4-keto group is critical for carotenoid binding. Further evidence of this is the fact that salinixanthol, an analogue of salinixanthin in which the 4-keto group is reduced to hydroxyl, does not bind and is not engaged in energy transfer. According to the crystal structure of xanthorhodopsin, the ring of salinixanthin in the binding site is turned out of the plane of the polyene conjugated chain. A similar conformation is expected for echinenone in the gloeobacter rhodopsin. We suggest that the 4-keto group in salinixanthin and echinenone allows for the twisted conformation of the ring around the C6-C7 bond and probably is engaged in an interaction that locks the carotenoid in the binding site.     

Mechanism of proton transport in bacteriorhodopsin from crystallographic structures of the K,L, M1, M2, and M2' intermediates of the photocycle

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.     

Characterization of metal ion-binding sites in bacteriorhodopsin

We have investigated the effects of the binding of various metal ions to cation-free bacteriorhodopsin ("blue membrane"). The following have been measured: shift of the absorption maximum from 603 to 558 nm (blue to purple transition), binding isotherms, the release of H+ upon binding, and the decay of the deprotonated intermediate of the photocycle, M412. We find that all cations of the lanthanide series, as well as the alkali and alkali earth metals earlier investigated, are able to bring about the absorption shift, whereas Hg2+ and Pt4+ are not. Sigmoidal spectroscopic titration curves and nonsigmoidal binding curves suggest that there are two high affinity sites for cations in bacteriorhodopsin. Binding to the site with the second highest affinity is responsible for the absorption shift. Divalent cation binding to blue membrane causes release of about six protons, whereas higher numbers of protons are released by trivalent cations, suggesting that the shift of absorption maximum involves proton release from carboxyl group(s). The metal ion bound to this site must be surrounded by carboxyl oxygen atoms acting together as a multidentate ligand with a specific geometry because multivalent ions are effective only when capable of octahedral coordination. Lanthanide ions dramatically inhibit M412 decay at pH above 6.3, an effect probably due to binding to lipid phosphoryl groups.     

基于遥感生态指数的道路网络生态效应分析——以福州市为例

以2000年和2016年两期Landsat影像为基础数据源,借助遥感生态指数（RSEI）对福州市生态环境进行评价,在此基础上,从道路缓冲区、城乡梯度带、剖面线三种不同取样方法定量探讨RSEI对路网的响应机制;再以500 m×500 m、1000 m×1000 m、1500 m×1500 m、2000 m×2000 m、2500 m×2500 m、3000 m×3000 m不同尺度的网格划分空间单元,运用全局空间自相关、地理加权回归分析等方法分析了道路核密度（KDE）和RSEI及其之间关系的空间异质性。结果表明：从2000年到2016年,福州市生态环境好的区域面积增幅大于生态环境差的区域面积,生态环境质量向好的方向发展。各类型道路缓冲区的RSEI变化规律都是呈从0 m到3000 m逐渐上升的趋势,其中国道、省道、县道、乡镇道路影响的阈值分别在900、900、450、750 m左右。在城乡梯度分析中,RSEI曲线的变化规律都是随着与行政中心距离的增大而增大,到达一定阈值后趋于平缓,甚至还有小幅度的下降,区级的影响阈值在20 km左右,县级的影响阈值在12 km左右;而KDE曲线的变化规律与RSEI相反,其变化阈值与RSEI正好对应。剖面线所经过的行政中心处,其RSEI为低值,KDE为高值,西北方向的内陆地区RSEI高于东南方向的沿海地区。在多尺度的地理加权回归分析中,1500 m×1500 m和2000 m×2000 m这两个网格单元采样下的空间集聚性较强,空间异质性明显,总体上来看,RSEI与KDE呈现负相关关系,且相关关系存在空间分异,负回归系数主要分布在研究区的中心区域。研究结果可为福州市生态建设和路网规划提供参考依据。     

Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.