嗜盐菌紫膜的质子泵功能

嗜盐菌生活在NaCl含量接近饱和的自然环境中。在缺氧和充分光照条件下,它的细胞膜能长出紫色的膜斑,称为紫膜,镶嵌在整个细胞膜上。在合适条件下,紫膜面积可占整个细胞膜表面的一半。紫膜上仅含有一种蛋白,叫细菌视紫质(bR,bacteriorhodopsin)。bR上的色素团——视黄醛(与哺乳动物视色素相同的一种色素团)通过质子化的席夫碱基与蛋白中赖氨酸的ε-氨基相连。bR分子在膜上排列极其     

细菌视紫红质中质子泵出的分子机理

细菌视紫红质(Bacteriorhodopsin,或bR)是盐生嗜盐菌(Halobacterium salinarium)等细菌的跨膜蛋白质,其色基视黄醛的光致异构化作用触发细菌视紫红质的一系列结构变化,把质子从细胞质泵到细胞外空间。对细菌视紫红质中质子泵出分子机理进行了描述。     

W86F/W86A突变对细菌视紫红质光循环影响的差异性分析

细菌视紫红质(bacteriorhodopsin,bR)是一种光驱受体蛋白,每个活性单元由3个单体组成,每个单体由一分子视蛋白和一分子的视黄醛发色团共价结合而成,其功能为从胞内向胞外定向传输质子,利用形成的质子浓度梯度将光能转化为化学能.光照后视黄醛发色团构型发生all-trans向13-cis转变,蛋白的构象也随之发生了一系列具有稳定中间态的变化并驱动质子的定向传递.为探讨bR视黄醛键合区保守性氨基酸色氨酸86 (Tryptop-han86,w86)对其光循环中间态和质子泵功能的影响,本研究采用定点突变技术将W86突变为侧链大小不同的F86和A86,通过原位紫外-可见光吸收光谱、闪光光解光谱、pH滴定、固体核磁共振等技术手段,探究W86F和W86A突变对bR光循环及质子泵功能影响差异性的分子机制.结果 表明,W86F和W86A突变均造成了bR暗适应状态下视黄醛顺反异构平衡向顺式构型占优的方向移动,且W86F突变可造成反式构型的完全消失.此外,无论是W86F还是W86A突变都造成了蛋白光循环中间态的减弱且衰减延长,以及质子泵功能减弱,但影响机制各有所不同,这可能与这两个突变对视黄醛多烯链上电子云的分布以及周围残基造成的扰动程度不同有关.     

N-溴代琥珀酰亚胺修饰菌紫质中色氨酸的光谱学研究

采用紫外-可见吸收光谱和荧光光谱方法研究了菌紫质（Bacteriorhodopsin,bR）中的8个色氨酸（Tryptophan,Trp）残基在被N-溴代琥珀酰亚胺（N-bromosuccinimide,NBS）修饰过程中的残基数目及对应的光谱变化。研究结果显示：随着NBS／bR摩尔比例增加逐渐被修饰的Trp残基有4个左右,如果NBS过量,则Trp残基的修饰个数最终可迭6～7个;伴随化学修饰出现Trp残基特征荧光峰值下降及峰位蓝移。研究结果揭示了bR中Trp残基可能的三种结构分布,对于进一步弄清bR中Trp-视黄醛（Retinal）偶联能量传递、单独Trp残基的荧光寿命和Tm残基在膜蛋白结构和功能中的作用具有积极而重要的意义。     

嗜盐菌中的类视紫红质蛋白

本文简要综述了在嗜盐菌中发现的四种含视黄醛的膜蛋白:bR,hR,sR-Ⅰ和sR-Ⅱ;着重介绍了对它们的结构和功能的研究状况,同时展示了这四种蛋白质在结构和功能方面的相似之处。     

M145F/F146M突变对光受体蛋白细菌视紫红质和古紫质4光循环的影响 *

古紫质4(archaerhodopsin 4,aR4)与细菌视紫质(bacteriorhodopsin,bR)同属于盐杆菌科,同源性59%,均为光驱质子泵。其功能是在光照条件下将质子由胞内泵到胞外形成跨膜质子梯度,该梯度差被膜上另外一种蛋白ATP合成酶用于ATP的合成,从而完成光能向生物能的转化。aR4和bR具有相似的光循环过程,但质子传递时序不同,aR4是先从胞内吸收质子再将质子释放到胞外,而bR恰好相反。甲硫氨酸-145是位于bR视黄醛发色团键合区的一个重要残基,对其光循环有着重要的影响,而在aR4中处在相应位置上的苯丙氨酸-146是其视黄醛键合区与bR唯一不相同的残基。因此通过定点突变,采用紫外可见吸收光谱、动力学光谱、质子泵功能检测、低温透射红外光谱等手段对比分析研究M145F和F146M单点突变对bR和aR4光循环造成的影响,有助于深入理解aR4结构与功能的关系。研究结果表明,M145F突变造成了bR光循环L的丢失和质子泵功能的减弱,而F146M突变并未对aR4的光循环造成显著影响,且突变后aR4质子释放时序没有反转,表明该位置上的残基在两个体系中的作用不尽相同。     

菌紫质分子的重组对结构及运动的影响

本实验用闪光诱导的瞬间二向色性方法分别测量了用全反视黄醛重组以及用四溴荧光素标记的菌紫质分子在脂质囊泡膜中的旋转扩散运动以及吸收过程随时间的变化(寿命).用全反视黄醛重组后不影响菌紫质分子在膜中的旋转扩散运动,它们与蜂毒的相互作用也无明显差别.用四溴荧光素标记的菌紫质分子的寿命很短,不可能用于测量菌紫质分子在膜中的旋转运动.     

细菌视紫红质蛋白的赖氨酸和丝氨酸残基在其膜环境中的自旋标记研究

用化学修饰和自旋标记ESR技术研究了bR中丝氨酸和赖氨酸残基的构象,结果表明PH对bR分子构象的影响是很明显的,在酸性条件下带有自旋探针的丝氨酸(Ⅰ-bR)和赖氨酸(Ⅱ-bR)的ESR波谱参数迥然不同,这与丝氨酸和赖氨酸残基位点微环境中的电荷效应有关.顺磁增宽剂能猝灭膜表面的自由基,而剩余的强固定化ESR信号显示出bR分子内部的‘刚性’构象.用2%Triton X—100处理可大大增加bR分子的‘柔性’,其ESR波谱特征与bR分子失去部分α螺旋结构和改变了它的某些运动方式有关.     

细菌视紫红质蛋白的赖氨酸和丝氨酸残基在其膜环境中的自旋标记研究

用化学修饰和自旋标记ESR技术研究了bR中丝氨酸和赖氨酸残基的构象,结果表明PH对bR分子构象的影响是很明显的,在酸性条件下带有自旋探针的丝氨酸(Ⅰ-bR)和赖氨酸(Ⅱ-bR)的ESR波谱参数迥然不同,这与丝氨酸和赖氨酸残基位点微环境中的电荷效应有关.顺磁增宽剂能猝灭膜表面的自由基,而剩余的强固定化ESR信号显示出bR分子内部的‘刚性’构象.用2%Triton X—100处理可大大增加bR分子的‘柔性’,其ESR波谱特征与bR分子失去部分α螺旋结构和改变了它的某些运动方式有关.     

温度对菌紫质结构转变的影响

测量了不同温度下菌紫质的紫外—可见及红外吸收光谱,比较天然与变性菌紫质的吸附等温线。证明菌紫质在80℃前的可逆结构转变表现为结晶晶格协同效应降低,部分芳香族氨基酸显露,蛋白质构象的微小变化使视黄醛微环境改变。100℃引起的结构改变为不可逆,此时菌紫质三、四级结构解体,分子链展开并重聚,二级结构也发生变化,出现β结构,同时还保留部出α-螺旋,视黄醛全部游离。并就菌紫质的高热稳定性能做了讨论。     

Light activation of the isomerization and deprotonation of the protonated Schiff base retinal

We perform an ab initio analysis of the photoisomerization of the protonated Schiff base of retinal (PSB-retinal) from 11-cis to 11-trans rotating the C10-C11=C12-C13 dihedral angle from 0° (cis) to -180° (trans). We find that the retinal molecule shows the lowest rotational barrier (0.22 eV) when its charge state is zero as compared to the barrier for the protonated molecule which is ∼0.89 eV. We conclude that rotation most likely takes place in the excited state of the deprotonated retinal. The addition of a proton creates a much larger barrier implying a switching behavior of retinal that might be useful for several applications in molecular electronics. All conformations of the retinal compound absorb in the green region with small shifts following the dihedral angle rotation; however, the Schiff base of retinal (SB-retinal) at trans-conformation absorbs in the violet region. The rotation of the dihedral angle around the C11=C12 π-bond affects the absorption energy of the retinal and the binding energy of the SB-retinal with the proton at the N-Schiff; the binding energy is slightly lower at the trans-SB-retinal than at other conformations of the retinal.     

Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: a theoretical analysis of structural elements

The transfer of a proton from the retinal Schiff base to the nearby Asp85 protein group is an essential step in the directional proton-pumping by bacteriorhodopsin. To avoid the wasteful back reprotonation of the Schiff base from Asp85, the protein must ensure that, following Schiff base deprotonation, the energy barrier for back proton-transfer from Asp85 to the Schiff base is larger than that for proton-transfer from the Schiff base to Asp85. Here, three structural elements that may contribute to suppressing the back proton-transfer from Asp85 to the Schiff base are investigated: (i) retinal twisting; (ii) hydrogen-bonding distances in the active site; and (iii) the number and location of internal water molecules. The impact of the pattern of bond twisting on the retinal deprotonation energy is dissected by performing an extensive set of quantum-mechanical calculations. Structural rearrangements in the active site, such as changes of the Thr89:Asp85 distance and relocation of water molecules hydrogen-bonding to the Asp85 acceptor group, may participate in the mechanism which ensures that following the transfer of the Schiff base proton to Asp85 the protein proceeds with the subsequent photocycle steps, and not with back proton transfer from Asp85 to the Schiff base.     

Characterisation of Schiff base and chromophore in green proteorhodopsin by solid-state NMR

The proteorhodopsin family consists of hundreds of homologous retinal containing membrane proteins found in bacteria in the photic zone of the oceans. They are colour tuned to their environment and act as light-driven proton pumps with a potential energetic and regulatory function. Precise structural details are still unknown. Here, the green proteorhodopsin variant has been selected for a chemical shift analysis of retinal and Schiff base by solid-state NMR. Our data show that the chromophore exists in mainly all-trans configuration in the proteorhodopsin ground state. The optical absorption maximum together with retinal and Schiff base chemical shifts indicate a strong interaction network between chromophore and opsin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mark Lorch and Andreas C. Woerner contributed equally to this work.     

Photochemical properties of a bacteriorhodopsin analog with 13-demethyl-13-trifluoromethyl retinal

It was shown that the substitution of the CF₃ group in the structure of retinal for the methyl group at C13 causes not only a decrease in the affinity of the proton for the nitrogen in the Schiff base (pK ～ 8.4) but also considerably changes the photochemical properties of the bacteriorhodopsin analog. At pH > 6.5, the rate of the Schiff base reprotonation during M decay depends on the proton concentration in the medium. In the photocycle of the yellow M-like form with the deprotonated Schiff base, a long-wavelength product absorbing at 625 nm is formed, which has a similar pH dependence of decay kinetics. The two processes also have similar activation energies (about 15 ± 1 kcal/mol). It is concluded that both cases involve proton transfer from an aqueous medium through the donor part of the channel to the Schiff base and Asp96, respectively. In the analog, however, the structure of water molecules necessary for the stabilization of the proton on the Schiff base is broken. As a result, dehydration of the preparation gives rise to a fraction of M-like form of bacteriorhodopsin with the deprotonated Schiff base.     

Photoreactions of bacteriorhodopsin

Bacteriorhodopsin is a membrane-bound light energy transducer which generates an electrochemical proton gradient. It undergoes a cyclic photoreaction in which five intermediates have been identified. During the cycle it releases a proton from one surface of the membrane and takes up a proton on the opposite surface. The active chromophore consists of retinal bound through a Schiff base to the protein. The Schiff base is deprotonized during the photoreaction cycle and appears to be involved in the transport of protons through the membrane. The retinal may also undergo an isomerization.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976This work was supported by NASA Grant NSG-7151 and NHLI Grant HL-06285     

Elimination of proton donor strongly affects directionality and efficiency of proton transport in ESR,a light-driven proton pump from Exiguobacterium sibiricum

ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H⁺ transfer.     

Thermodynamics of proton transfer in carboxylic acid-retinal Schiff base hydrogen bonds with large proton polarizability

During the photocycle of bacteriorhodopsin (BR) the chromophore, a retinal Schiff base, is deprotonated. Simultaneously an asp residue is protonated. These results suggest that this deprotonation occurs via a Schiff base - asp hydrogen bond. Therefore, we studied carboxylic acid - retinal Schiff base model systems in CCl4 using IR spectroscopy. The IR spectra show that double minimum proton potentials are present in the OH ... N in equilibrium with O- ... HN+ H-bonds formed and that the proton can easily be shifted in these bonds by local electrical fields. The thermodynamic data of H-bond formation and proton transfer within these H-bonds are determined. On the basis of these data a hypothesis is developed with regard to the molecular mechanism of the deprotonation of the Schiff base of BR.     

Atomic resolution structures of bacteriorhodopsin photocycle intermediates: the role of discrete water molecules in the function of this light-driven ion pump

High-resolution X-ray crystallographic studies of bacteriorhodopsin have tremendously advanced our understanding of this light-driven ion pump during the last 2 years, and emphasized the crucial role of discrete internal water molecules in the pump cycle. 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 via water 402 and the initial proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-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 in the otherwise very hydrophobic region between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The M intermediate trapped in the D96N mutant corresponds to a late M state in the transport cycle, after protonation of Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. The M intermediate from the E204Q mutant corresponds to an earlier M, as in this mutant the Schiff base deprotonates without proton release. The structures of these two M states reveal progressive displacements of the retinal, main chain and side chains induced by photoisomerization of the retinal to 13-cis,15-anti, and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pK(a)s of the Schiff base, Asp85, the proton release group and Asp96. The structure for the M state from E204Q suggests, moreover, that relaxation of the steric conflicts of the distorted 13-cis,15-anti retinal plays a critical role in the reprotonation of the Schiff base by Asp96. Two additional waters now connect Asp96 to the carbonyl of residue 216, in what appears to be the beginning of a hydrogen-bonded chain that would later extend to the retinal Schiff base. Based on the ground state and M intermediate structures, models of the molecular events in the early part of the photocycle are presented, including a novel model which proposes that bacteriorhodopsin pumps hydroxide (OH(-)) ions from the extracellular to the cytoplasmic side.     

Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK_a of the Schiff base (the primary proton donor) and the low pK_a of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

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.