Effects of mutations of Lys41 and Asp102 of bacteriorhodopsin

Bacteriorhodopsin (BR) is a retinal protein that functions as a light-driven proton pump. In this study, six novel mutants including K41E and D102K, were obtained to verify or rule out the possibility that residues Lys41 and Asp102 are determinants of the time order of proton release and uptake, because we found that the order was reversed in another retinal protein archaerhodopsin 4 (AR4), which had different 41th and 102th residues. Our results rule out that possibility and confirm that the pK(a) of the proton release complex (PRC) determines the time order. Nevertheless, mutations, especially D102K, were found to affect the kinetics of proton uptake substantially and the pK(a) of Asp96. Compared to the wild-type BR (BR-WT), the decay of the M intermediate and proton uptake in the photocycle was slowed about 3-fold in D102K. Hence those residues might be involved in proton uptake and delivery to the internal proton donor.     

菌紫质的结构和功能研究进展

紫膜中具有质子泵功能的菌紫质(bR)是整合膜蛋白,它是7个α螺旋跨膜蛋白家族的基本原型.目前,具有光驱动质子泵的bR是最典型的高效离子转运蛋白之一.它很可能成为其载体转运机制在分子甚至原子水平上被阐明的第一个膜蛋白.概述了近年来对菌紫质结构,光循环和质子泵机理研究的进展.     

菌紫质研究的新进展

菌紫质(BR)是嗜盐菌紫膜中的唯一蛋白质,野生型的BR分子含有248个氨基酸残基,其中一个视黄醛通过希夫碱基连结在第216位赖氨酸上,它具有质子泵的功能．光照下,BR进行光循环,光循环又与质子泵过程相关联．菌紫质的结构和功能方面的研究已有很大进展,但其光循环途径和质子泵的机理还不太清楚．文章概述了近年来对菌紫质结构,光循环和质子泵机理研究的进展,尤其对争论较大的菌紫质光循环途径的四类模型作了较详细的介绍．     

细菌视紫红质的质子传输机理

细菌视紫红质（bR）是嗜盐菌紫膜中的唯一蛋白质成分, 具有质子泵、电荷分离和光致变色功能. bR分子中的发色团视黄醛通过质子化席夫碱以共价键与Lys216相连. bR分子受可见光照射后, 视黄醛发生从全-反到13-顺式构型的异构化, 导致席夫碱的去质子化,继之以可极化基团位置的改变. 力场的变化引起包括蛋白质三级结构在内的诸多变化, 这些变化促进并保证了质子从细胞质侧向细胞外侧的定向传输.     

细菌视紫红质的光电响应特性和机制

在ITO导电玻璃上制备定向细菌视紫红质 (BR)电泳沉积膜或LB膜组成光电池系统 ,在短脉冲激光照射下 ,测定其脉冲响应光电压 ;在间断光照射下 ,测定其对光强变化产生的微分响应信号。对脉冲光电响应和微分响应的机理及其关系进行理论分析和解释 ,认为脉冲响应是BR分子内部生色团快速光极化引起的电荷分离和希夫碱及其周围氨基酸去质子化和再质子化过程引起的质子定向运输产生的位移电流 ,是一个快反应过程 ,是微分响应的早期反应和基础。微分响应则是由于菌紫质的光驱动质子泵产生的连续质子流在光开和光关瞬间引起光电池系统充放电以及测量电路的耦合特性引起的 ,是一个慢变化过程     

细菌视紫红质 E204Q 和 I119T/T121S/A126T突变体的构建及其功能研究

应用定点突变的方法获得了细菌视紫红质 (bacteriorhodopsin , BR) 的单突变体 BR_E204Q和三突变体 BR_{I119T/T121S/A126T}. 通过功能研究发现, BR 蛋白的单突变体 BR_E204Q的 M 态寿命为 7.10 ms ,三突变体 BR_{I119T/T121S/A126T}的 M 态寿命为 8.23 ms ,均较野生型 BR 蛋白 (6.23ms) 有所延长,三突变体表现得更为显著,其 M 态延长时间可超出野生型的 32％. 同时单突变体 BR_E204Q和三突变体 BR_{I119T/T121S/A126T}的质子泵功能也均有改变,都比野生型 BR 蛋白有所下降,其中三突变体 BR_{I119T/T121S/A126T}下降得更为明显.     

Photoelectrochemical Cycle of Bacteriorhodopsin

The scheme of the bacteriorhodopsin photocycle associated with a transmembrane proton transfer and electrogenesis is considered. The role of conformational changes in the polypeptide chain during the proton transport is discussed.     

X-Ray Crystallography of Bacteriorhodopsin and Its Photointermediates: Insights into the Mechanism of Proton Transport

In the last few years, detailed structural information from high-resolution x-ray diffraction has been added to the already large body of spectroscopic and mutational data on the bacteriorhodopsin proton transport cycle. Although there are still many gaps, it is now possible to reconstruct the main events in the translocation of the proton and how they are coupled to the photoisomerization of the retinal chromophore. Future structural work will concentrate on describing the details of the individual proton transfer steps during the photocycle.     

Proton Transport Mechanism of Bacteriorhodopsin as Revealed by Site-Specific Mutagenesis and Protein Sequence Variability

A large share of the current ideas about the mechanism of proton transport by bacteriorhodopsin has emerged from studies of site-specific mutants. This review is an attempt to check some of these ideas against the natural variability in the primary structure of the protein.     

Bacteriorhodopsin. Correspondence of the photocycle and electrogenesis with sites of the molecule

Correspondence of phases of electrogenesis, photocycle transitions, and proton transfer with the proton transporting groups of bacteriorhodopsin was studied. The structure of bacteriorhodopsin was considered by the file 1c3w and projections of sites of the proton movement pathway onto the normal to the purple membrane were measured. The dielectric permeability of the terminal site of the semichannel Schiff base external surface of the purple membrane was noticeably higher than in the center of the membrane.Translated from Biokhimiya, Vol. 69, No. 12, 2004, pp. 1725–1728.Original Russian Text Copyright © 2004 by Khitrina, Ksenofontov.     

脉冲激光对细菌视紫红质的瞬态光电荷转移动力学研究

细菌视紫红质（ＢＲ）是一种可进行光能存储与转换的蛋白质分子,在光作用下,能极快地产生电荷分离与电光响应信号。实验采用自制的波长可调的染料激光,在μｓ－ ｍ ｓ时域内对ＢＲ的动态光电响应信号进行了研究,总结了当激发脉冲光波长改变或强度改变时光电压变化的规律,并定性地分析了ＢＲ产生上述现象的机理。     

Radiation Chemistry of Foods

An intermediate radical, ?H₂OH, was produced in aqueous methanol solution containing nitrous oxide by γ-irradiation. Yields of ethylene glycol and formaldehyde, the major and the minor product from ?H₂OH, respectively, changed on the addition of some solutes. Cysteine lowered the both product yields to zero even at a low concentration of 5 × 10^?5m. Oxygen of low concentrations (2.5~7.5 × 10^?5 m) changed effectively the major product from ethylene glycol to formaldehyde. k _(CySH+?H2OH)/k_(O2+?H2OH) was calculated as 0.5.

Ascorbic acid (5 × 10^?5 m) lowered ethylene glycol yield to 48%, cystine (10^?3m) to 15%, methionine (10^?3m) to 31%, histidine (10^?3m) to 42%, tryptophan (10^?3m) 46%, tyrosine (10^?3m) to 77%, phenylalanine (10^?3m) to 73%, hypoxanthine (10^?3m) to 37%, adenine (10^?3m) to 52%, uracil (10^?3m) to 20%, thymine (10^?3m) to 10%, cytosine (10^?3 m) to 49%, rutin (10^?3m) to 23%, pyrogallol (10^?3m) to 41%, and gallic acid (10^?3m) to 78% of the control. These results suggest that the reactions of the secondary radicals such as ?H₂OH perform an important role in material change of foods irradiated with γ rays.     

Crystal structures of acid blue and alkaline purple forms of bacteriorhodopsin

Bacteriorhodopsin, a light-driven proton pump found in the purple membrane of Halobacterium salinarum, exhibits purple at neutral pH but its color is sensitive to pH. Here, structures are reported for an acid blue form and an alkaline purple form of wild-type bacteriorhodopsin. When the P622 crystal prepared at pH 5.2 was acidified with sulfuric acid, its color turned to blue with a pKa of 3.5 and a Hill coefficient of 2. Diffraction data at pH 2-5 indicated that the purple-to-blue transition accompanies a large structural change in the proton release channel; i.e. the extracellular half of helix C moves towards helix G, narrowing the proton release channel and expelling a water molecule from a micro-cavity in the vicinity of the retinal Schiff base. In this respect, the acid-induced structural change resembles the structural change observed upon formation of the M intermediate. But, the acid blue form contains a sulfate ion in a site(s) near Arg82 that is created by re-orientations of the carboxyl groups of Glu194 and Glu204, residues comprising the proton release complex. This result suggests that proton uptake by the proton release complex evokes the anion binding, which in turn induces protonation of Asp85, a key residue regulating the absorption spectrum of the chromophore. Interestingly, a pronounced structural change in the proton release complex was also observed at high pH; i.e. re-orientation of Glu194 towards Tyr83 was found to take place at around pH 10. This alkaline transition is suggested to be accompanied by proton release from the proton release complex and responsible for rapid formation of the M intermediate at high pH.     

Unraveling the mechanism of proton translocation in the extracellular half‐channel of bacteriorhodopsin

Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long‐range proton translocation between the central (the retinal Schiff base SB216, D85, and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation states of the SB216, D85, D212, and E204. The side‐chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side‐chain reorientation of R82 modulates the hydrogen‐bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton‐transfer in the methyl guanidinium‐hydronium‐hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the bacteriorhodopsin models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O′ state where the proton on D85 is transferred to D212. Proteins 2016; 84:639–654. © 2016 Wiley Periodicals, Inc.     

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。     

细菌视紫红质的两种光电微分响应及其机制

利用MATLAB软件对细菌视紫红质 (BR)膜光电器件的脉冲响应实验数据进行拟合 ,得到器件的冲激响应函数。据此用SIMULINK模块构造出了反映BR光电器件特性的仿真系统。利用此系统对不同间断光入射BR光电器件时的输出响应信号进行了仿真计算。通过分析得出结论 :以前所描述的微分响应 (发生在毫秒到秒的时间量级 ,在光打开时产生一个正脉冲 ,在光关掉时产生一个负脉冲 )并非BR分子固有的特性 ,部分是由于测量电路引起的。BR分子本身特性引起的微分响应是发生在微秒时间量级 ,而且在光打开时产生一个负脉冲 ,在光关掉时产生一个正脉冲。对这两种微分响应产生的机制分别进行了探讨     

Regeneration of Bacteriorhodopsin from Thermally Unfolded Bacterio-Opsin and All-trans Retinal at High Temperatures

The temperature dependence of regeneration of bacteriorhodopsin (bR) from its apoprotein, bacterio-opsin (bO), and all-trans retinal was investigated using two different procedures to probe the structural properties of bO at high temperatures. Regeneration experiments performed at 25 °C after incubation of bO within the temperature range of 35–75 °C indicate that irreversible thermal unfolding begins at 50 °C. When bO is incubated for one hour and mixed with retinal at the same elevated temperatures, however, a greater extent of regeneration to bR occurs, even at temperatures ranging from 50 to 65 °C. These experimental results indicate that regeneration of bR occurs from thermally unfolded bO and suggest dynamic structural fluctuation of bO in the unfolded state.     

Effects of Arg26 and Lys27 mutation on the bioactivity of HNTX-IV

Hainantoxin-IV (HNTX-IV) was isolated from the Chinese bird spider Ornithoctorcs hainana and identified as a novel antagonist of tetrodotoxin-sensitive (TTX-S) sodium channels. As revealed by the solution structure of HNTX-IV solved by two-dimensional nuclear magnetic resonance (2D-NMR), HNTX-IV adopts an inhibitor cystine knot motif. To check the role of basic residues during HNTX-IV’s interaction with TTX-S sodium channels, R26A and K27A mutants of HNTX-IV were constructed by solid-phase chemical synthesis. The synthesized peptides were purified and refolded under optimized oxidation conditions. Correct synthesis and folding were confirmed by MALDI-TOF mass spectrometry and NMR spectroscopy, respectively. Using the whole-cell patch-clamp technique, Lys27 but not Arg26 was identified as a key residue for HNTX-IV’s bioactivity against TTX-S sodium channels, because R26A-HNTX-IV showed slightly reduced activity and K27A-HNTX-IV showed almost no inhibition. Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(4): 499–503 [译自: 中国生物化学与分子生物学报]     

Importance of Lipids for Bacteriorhodopsin Structure, Photocycle, and Function

This review begins with a brief history of early studies on the involvement of lipids in certain bacteriorhodopsin (BR) properties. Such properties include the regulation of the pK for the purple to blue transition caused by deionization, and the reformation of trimers from monomers after exposure of the purple membrane to Triton X-100. Most of the review is devoted to newer studies which indicate an important role for the neutral lipid squalene in the functional stability of the fast-decaying M-intermediate, for its decay through a pathway involving the O-intermediate, and for the regulation of the relative amounts of slow-decaying and fast-decaying forms of M. Participation of a peripheral acidic amino acid in the overall expression of fast-decaying M is also discussed. Initial studies suggest that the acidic amino acid may be Asp36 and/or Asp38.