Histological and Physiological Investigation of Channelrhodopsin-2 and Halorhodopsin in the Dorsal Cochlear Nucleus

We have delivered viral vectors containing either Chop2 fused with GFP, Channelrhodopsin-2 （ChR2）, or Halorhodopsin （HaloR） fused with mCherry （to form light gated cation channels or chloride pumps, respectively）, into the dorsal cochlear nucleus （DCN）. One to eighteen months later we examined the CN and inferior colliculus （IC） for evidence of virally transfected cells and processes. Production of ChR2 and HaloR was observed throughout the DCN. Rhodopsin localization within neurons was determined, with elongate, fusiform and giant cells identified based on morphology and location within the DCN. Production of ChR2 and HaloR was found at both the injection site as well as in regions projecting to and from the DCN. Light driven neuronal activity in the DCN was dependent upon the wavelength and intensity of the light, with only the appropriate wavelength resulting in activation and higher intensity light resulting in more neuronal activity. Transfecting cells via viral delivery of rhodopsins can be useful as a tract tracer and as a neuronal marker to delineate pathways. In the future rhodopsin delivery and activation may be developed as an alternative to electrical stimulation of neurons.

CONSTRUCTION AND EXPRESSION OF BACTERIORHODOPSIN MUTANT IN HALOBACTERIUM HALOBIUM

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Studies on HEPES Stabilizing M Intermediate of Bacteriorhodopsin

EPES has proved to stabilize M intermediate of Bacteriorhodopsin. Absorp-tion and Raman spectra showed that there was a more stable form of M intermedi-ate in BR-HEPES solution. It was found by SPS that there were two main interme-diates . M and O , existing in BR-HEPES solution.

The behaviours of optical novelty filter based on bacteriorhodopsin film

The quality of the novelty filter image is investigated at different intensities of the incident blue and yellow beams irradiating a bacteriorhodopsin （bR） film. The relationship between the transmitted blue beams and the incident yellow beams is established. The results show that the contrast of the novelty filter image depends on the lifetime of longest lived photochemical state （M state）. These results enable one to identify the direction of a moving object and to improve the quality of the novel filter image by prolonging the lifetime of M state.

The 3rd-order nonlinearity of bacteriorhodopsin by four-wave mixing

The 3rd-order nonlinear optical susceptibility x(3) and the response time of the light-transducing biomolecule bacteriorhodopsin were measured with the four-wave mixing technique and a picosecond frequency-doubled Nd: YAG laser (532 nm). The x(3) and the response time measured are 10-9 esu and 20 ps , respectively. The possible mechanism for generating the 3rd-order nonlinear optical susceptibility x(3) and response time were discussed.

Electron microscopic observation and rotational diffusion measurement of bacteriorhodopsin in lipid vesicles

The morphology of bacteriorhodopsin reconstituted into dimyristoylphosphatidylcholine and egg-phosphatidylcholine vesicles was observed by freeze-fracture electron microscopy. The rotational diffusion of bacteriorhodopsin at different concentrations of melittin was measured by observing flash-induced transient dichroism in dimyristoylphosphatidylcholine vesicles. In the presence of melittin, bacteriorhodopsin molecules in dimyristoylphosphatidylcholine vesicles were aggregated into large particles or patches, and the ability of rotational diffusion of bacteriorhodopsin in vesicles was decreased. This suggests that melittin produces its effect via direct electrostatic interaction with bacteriorhodopsin. Low temperature-induced aggregation of bacteriorhodopsin was also observed in dimyristoylphosphatidylcholine vesicles. Low temperature may cause phase separation. Bacteriorhodopsin was also successfully reconstituted into egg-phosphatidylcholine vesicles, but low temperature-induced aggregation of

Imaging bacteriorhodopsin-like molecules of claret-membranes from Tibet halobacteria xz515 by atomic force microscope

Halobacteria H.sp.xz 515 was isolated from a salt lake in Tibet. Although proton release-and-uptake across claret membrane is in reverse order compared to bacteri-orhodopsin in purple membrane from Halobacterium Sali-narum, and its efficiency of proton pump is much lower, AFM image shows that the molecules are still arranged in a two-dimensional hexagonal lattice of trimers. Primary structure of C- to G-helix of the archaerhodopsin shows that it has only 56% homology with bacteriorhodopsin. But the interactive amino acid residues at the interface between B-and D-helixes are conserved. These amino acid residues are believed to play a significant role in the stability of protein oligomers.

The dependence of pH and ionic strength of kinetic absorption spectroscopy of acetylated bacteriorhodopsin

Bacteriorhodopsin （bR） in purple membrane （PM） from H. Halobium is a light-drivenproton pump. Its seven transmembrane helices form an internal proton channel. Anall-trans retinal covalently links up via protonated Schiff base with K216 inside thechannel. Upon illumination, the retinal isomerization around the C13-C14 double bond drivesprotons translocation through the channel from the cytoplasm into the medium, and

pH-dependence of interaction between melittin and bacteriorhodopsin

Melittin differentially slowed down the fast (M412f) and the slow (M412s) decay components of the photocyde intermediate M of trimeric bacteriorhodopsin in purple membrane while it accelerated the M412s of Triton X-100-solubilized bacteriorhodopsin monomers. Raising the bulk pH could enhance the effect of melittin on the M412s of bacteriorhodopsin in these two states. From pH 5.5 to 8.8, melittin slightly influenced the yield of intermediate M in purple membrane, whereas the yield of M412s decreased and subsequently reversed with the addition of melittin. Moreover, the monomeric bacteriorhodopsin bleached more readily in the presence of melittin and the higher pH made the bleaching effect of melittin more intensive as well. These results re-certify our former suggestions that there was electrostatic interaction between melittin and bacteriorhodopsin, and indicate that the biphasic M decay may not result from the well-known linear kinetic scheme (M→N →BR). At last the mechanisms underlying the

Laser-induced bacteriorhodopsin LB films'fast photoelectric dynamics

Ten layers of oriented bacteriorhodopsin (BR) LB films are prepared on ITO conductive glass toconstitute an ITO electrode/BR LB films/KCl solution/Cu electrode wet photocell.A Q-switched double frequency Nd:YAG laser with 10 Hz repetitive rate and 8 ns pulse width is used to excite the BR LB films.The photoelectric signal is directly captured by a 300 MHz digitizing oscilloscope.A theory on the BR photoelectric dynamics is established.By nonlinear least square fitting of the experimental curves acquired at different time bases,the experimental equation corresponding to the theoretical model is obtained,and the charge displacements and the decay time constants for each step in the BR photocycle are calculated.

PHOTOCHEMICAL INTERACTIONS BETWEEN NITROXIDE FREE RADICALS AND BACTERIORHODOPSIN IN SOLUTION

The purple membrane found in Halobacterium halobium has been shown to contain only a single protein, bacteriorhodopsin (bR), which functions as a light-driven proton pump. The visible absorption of bR is a result of the presence of a retinal chromophore.

Photovoltage of Bacteriorhodopsin in the Purple Membrane Langmuir-Blodgett Film

With the rise of studying biomolecular electronics and biocomputer since the 1980s, bactefiorhodopsin (BR) of purple membrane (PM) from Halobacteria halobium has become an outstanding candidate for studying biomolecular devices due to its stable structure, light-driven proton pumping capability, bistable property, photoelectric responses from ps to ms, as well as its potential application to developing biomolecular devices, biochips and so on. However, the BR not only must, but also can be oriented due

Photocycle of 13-cis-retinal in bacteriorhodopsin caused by destruction to the cooperative effect of purple membrane

The steady absorption and kinetic changes of M 412 intermediate of the light and dark adapted bacteriorhodopsin (BR) solubilized by different concentrated Triton X 100 were investigated. The results indicated that the cooperative effect existing within the trimeric BR of native purple membrane (PM) was damaged in the system containing the surfactant since the component and structure of the bilayer lipid membrane in PM varied due to the solubilization of partial PM lipids by Triton X 100. The destruction to the cooperative effect of BR ultimately caused 13 cis retinal of the dark adapted BR to take part in BR photocycle and also to generate the deprotonated M 412 intermediate.

Preparation of a gene-engineering mutant of bacteriorhodopsin BR-D96V and corresponding poly(vinyl alcohol)-based functional composite films

Bacteriorhodopsin(BR)exhibits,as a membrane protein in Halobacterium salinarum,unique photoresponsive behaviors,and shows promise as a functional information material.A new mutant of BR with the 96th aspartic acid replaced by valine(BR-D96V)was obtained and then a composite film of BR-D96V in a synthetic polymer matrix was prepared in this research.The mutant BR-D96V was expressed in a bacterio-opsin deficient halobacterial strain(L33)by gene engineering.Although valine is very hydrophobic,this point mutant keeps the basic biological activities,namely,photoelectric and photochromic responses.Nevertheless,the lifetime of M intermediate in the BR mutant is nearly two orders of magnitude longer than that of wild-type BR in neutral aqueous solution,which benefits its potential application as an information material.The M lifetime is further significantly prolonged after embedding BR-D96V into poly(vinyl alcohol)(PVA).It was also found that BR-D96V is very sensitive to water content in comparison with wild-type BR and another BR mutant.

Photoelectrochemical Properties of Bacteriorhodopsin Langmuir-Blodgett Films on ITO Conductive Electrode

Langmuir-Blodgett films containing bacteriorhodopsin were deposited on ITO conduc-tive electrodes.A sandwiched photocell with a junction structure of ITO/bR/electrolyte/ITO has beenconstructed,in which the bR LB film was directly put into contact with an aqueous electrolyte immobi-lized in an agar gel.Under visible light irradiation,the photocell produced a transient photocurrent due tothe change of light intensity,which characterized vision imitative material.A photoalarm device based ondifferential responsivity phenomena was fabricated.

Effects of Cholate on the Stability of Bacteriorhodopsin in Alkaline Solution

The effects of cholate on the structure of bacteriorhodopsin (bR) in the purple membrane and the effects of alkali on native and cholate treated bR were studied using absorption spectra, fluorescence spectra and CD spectra. Treatment mass fraction of 10% cholate at room temperature for 48 h had no obvious effect on the structure of bR, yet changed the composition of the purple membrane, making cholate treated bR more stable in alkaline solution than native bR.

Tunable photocycle kinetics of a hybrid bacteriorhodopsin/quantum dot system

The inclusion of inorganic nanoparticles in biological environments has led to the creation of hybrid nanosystems that are employed in a variety of applications. One such system includes quantum dots (QDs) coupled with the photoactive protein, bacteriorhodopsin (BR), which has been explored in developing enhanced photovoltaic devices. In this work, we have discovered that the kinetics of the BR photocycle can be manipulated using CdSe/CdS (core/shell) QDs. The photocycle lifetime of protein samples with varying QD amounts were monitored using time-resolved absorption spectroscopy. Concentration-dependent elongations of the bR and M state lifetimes were observed in the kinetic traces, thus suggesting that excitonic coupling occurs between BR and QDs. We propose that the pairing of BR with QDs has the potential to be utilized in protein-based computing applications, specifically for real-time holographic processors, which depend on the temporal dynamics of the bR and M photointermediates.

亮氨酸94在光驱质子泵古紫质-4中的功能

利用基因定点突变技术分别将bR/aR4(细菌视紫红质/古紫质-4)中L93/L94(亮氨酸93/亮氨酸94)突变为侧链不同的丙氨酸、苯丙氨酸和极性不同的苏氨酸,得到bR、含菌红素BI-aR4和缺失菌红素BE-aR4体系突变蛋白,采用紫外-可见光吸收光谱、闪光光解光谱技术和pH滴定等方法,对比研究L94在aR4中对视黄醛构象、光循环及质子传输的影响及其同菌红素的关系。结果表明,aR4中L94同样会通过空间位阻效应调控视黄醛键合区构象和质子通道的螺旋分布,以维持视黄醛顺-反异构平衡和再异构化过程,并且促进质子的释放,保证蛋白高效的质子泵活性。此外,L94对aR4结构和功能的影响会受到菌红素的调制,在缺失菌红素的情况下L94突变对aR4螺旋的扰动更加剧烈,导致质子供体和质子释放簇周围的氢键网络发生变化。

嗜盐古菌中氯视紫红质基因序列的遗传分析

采用PCR方法扩增了嗜盐古菌AB19、AB30和AJ5编码螺旋C至螺旋G的氯视紫红质(halorhodopsin,HR)蛋白基因片断和16S rRNA基因(16S rDNA),测定了它们的核苷酸序列,与已报道的hr基因序列差异显著.获得AJ5 HR蛋白基因序列在Halobiforma属中尚属首次.对hr基因序列进行的遗传分析,包括GC含量、转换与颠换的比值、密码子使用偏好,表明hr基因面临进化选择和偏倚突变压的双重制约,是一类进化程度较高的基因.对比br基因编码蛋白螺旋C到G的序列发现它们具有相似的种属特征.采用BR和HR蛋白螺旋C到G序列构建系统发生树比传统的16S rDNA序列具有优势,BR和HR可以作为两种新型的分子指标.

br基因的克隆及表达

细菌视紫红质 ( Bacteriorhodopsin,BR)是嗜盐菌细胞膜上唯一的一种光能转换色素蛋白 ,BR以视黄醛 ( retinal)作辅基 ,在光诱导下吸收光子后会产生一系列的光循环中间体 ,最后又回到原始状态 .由于 BR这种独特的分子结构和光循环过程 ,及其作为一种具有光敏特性的蛋白质生物分子 ,可通过生物学技术进行改造 ,正引起光子学研究及生物学研究领域的高度注意 .从 Genbank查询 br基因的全序列 ,然后采用聚合酶链反应 ( Polymerase chain reaction,PCR)技术 ,采用 DNA star软件辅助设计了上下游引物 ,以嗜盐菌 ( H alobactrium H alobium)的基因组 DNA为模板克隆获得了完整的细菌视紫红质基因 br,并且将经测序鉴定的 br基因重组到原核高效表达载体 p GEX- 4 T- 2中 ,转化大肠杆菌 E.coli.,将阳性转化子经 IPTG诱导表达获得 BR与 GST(谷胱苷肽转移酶 )的融合蛋白后 ,提取蛋白进行 SDS-PAGE电泳、Western印迹反应 ,检测表达结果为阳性 ,且表达蛋白的大小与预想的一致 .