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.     

用半合成方法证明B_(22)-精氨酸不是胰岛素表现活性所必需的

胰岛素六甲酯经胰蛋白酶和羧肽酶B水解,得去B链羧端九肽(B_(22-30))胰岛素五甲酯的两个α-氨基用叔丁氧羰酰(BOC)保护后与X-D—Ala—PheOMe(X=Arg,D-Arg,Gly,Lys或Asp)缩合,去除所有的保护基后得B_(22)改变的去B链羧端六肽胰岛素(D-Ala~B_(22)-DHI)。这些类似物的生物活性表明:Arg~B_(22)能为Lys或Asp代替,这结果说明Arg~B_(22)不是胰岛素活力所必需。但Arg~B_(22)若为D-Arg或Gly代替,活力很低。     

用半合成方法证明B_(22)-精氨酸不是胰岛素表现活性所必需的

胰岛素六甲酯经胰蛋白酶和羧肽酶B水解,得去B链羧端九肽(B_(22-30))胰岛素五甲酯OMe—(DNIOH)。OMe—DNIOH的两个α-氨基用叔丁氧羰酰(BOC)保护后与X-D-Ala-PheOMe(X=Arg,D-Arg,Gly,BOG—Lys或OTB—Asp)缩合,去除所有的保护基后得B_(22)改变的去B链羧端六肽胰岛素(D-Ala~(B_(22))-DHI)。这些类似物的生物活性表明:Arg~(B_(22)能为Lys或Asp代替,这结果说明Arg~(B_(22))不是胰岛素活力所必需。但Arg~(B_(22))若为D-Arg或Giy代替,活力很低。     

野生与豢养长江江豚血清氨基酸含量比较

应用高效液相色谱(HPLC)技术,首次测定了湖北石首长江天鹅州白豚自然保护区野生长江江豚(Neophocaena phocaenoides asiaeorientalis)和中国科学院水生生物研究所白豚馆人工饲养的长江江豚血清中17种氨基酸的含量.结果表明,除了脯氨酸Pro、蛋氨酸Met和组氨酸His外,人工饲养江豚血清中其余14种氨基酸(天门冬氨酸Asp、谷氨酸Glu、丝氨酸Ser、精氨酸Arg、甘氨酸Gly、苏氨酸Thr、丙氨酸Ala、异亮氨酸Ile、亮氨酸Leu、苯丙氨酸Phe、缬氨酸Val、赖氨酸Lys、酪氨酸Tyr、胱氨酸Cys)的含量显著高于野生长江江豚血清中相应氨基酸的含量.野生江豚和人工饲养江豚的血清氨基酸含量均没有显著的性别差异.野生江豚性成熟个体与未成熟个体之间血清氨基酸含量也没有显著性的差异.在所检测的17种氨基酸中,豢养江豚Glu含量最高,其次为Asp和Lys.野生江豚同样是Glu最高,其次是Lys和Asp.豢养和野生江豚都是Met含量最低.野生和豢养江豚必需氨基酸(EAA)和非必需氨基酸(NEAA)之间的比率分别是0.83和0.92,具有极显著的差异(p <0.01).     

血清胸腺因子的固相合成及抗血清的制备

通过Bpoc·Ser(tBu)·Asp·OtBu的β-羧基上树脂和依次用Fmoc·Gly·Gly·OHFmoc·Gln·ONP,Fmoc.Ser(tBu).OH,Fmoc.Lys(Boc)·OH,Fmos·Ala·OH及pGlu·OH伸长,接着氨解和酸解的策略,我们用固相方法合成了血清胸腺因子(FTS,Thymulin),pGlu·Ala·Lys·Ser·Gln·Gly·Gly·Ser·Asn;制备了具有最大生物活性的该因子和Zn~(2+)的复合物,FTS-Zn;用该复合物与牛血清白蛋白(BSA)结合所形成的免疫原免疫家兔,获得了抗FTS-Zn~(++)的抗血清,通过酶联免疫吸附分析法(ELISA)测得其滴度高达1:12800。     

一种重组抗胰蛋白酶单体人胰岛素突变体的设计、制备和鉴定

用缺口双链DNA的定向突变方法分别将胰岛素前体中B链第 2 2、2 8、2 9和 3 0位改变为Asp、Lys、Pro和Lys,酵母分泌表达的前体经胰蛋白酶直接酶切 ,得到重组 [B2 2Asp、B2 8Lys、B2 9Pro、B3 0Lys]人胰岛素。它与受体的结合能力约为猪胰岛素的 6% ,而体内生物活力保留 5 0 %。通过FPLC分子筛测定其自身结合能力 ,在生理条件下浓度达 10 -4mol/L时它以单体形式存在。作为可抗胰蛋白酶酶解的单体胰岛素类似物 ,它可能具有一定的应用前景     

牛肝辅酶Ⅱ依赖性视黄醇脱氢酶cDNA的克隆及组织表达

迄今为止的研究证明 ,维生素A亦称视黄醇(retinol)的生理功能是通过其两步氧化代谢产物视黄醛与视黄酸 (亦称维甲酸 )来完成的 .视黄醛通过其光学异构体 1 1 顺式视黄醛与视觉细胞内的视蛋白 (opsin)结合组成视色素 .感光时 ,1 1 顺式视黄醛转变成全反式视黄醛从视蛋白脱落 ,这一过程同时传导到大脑产生视觉[1 ] .全反式维甲酸 (all transretinoicacid)则通过与其在核内受体 (RARα ,β ,γ)结合调节基因的转录来发挥其许多重要的生理功能 ,包括正常胚胎的发育 ,形态、神经系统的形成 ,成体动物的生长、发育、繁殖等 ,并通过调解组织及…     

截短型细胞毒素Gelonin的分子构象和生物活性

为了探索免疫络合物中具杀伤靶细胞的毒素,gelonin的结构与功能的关系,根据化学合成的gelonin基因序列和3维分子构象设计了N端区Gly,Leu,Asp和/或C端区Asp,Lys,Asp,Pro,Lys缺失的gelonin. 以重组质粒pE gel为模板,在相应引物存在下,用PCR法获得5′端区和/或3′端区碱基序列缺失的gelonin基因片段. 经克隆、表达和纯化得到3种截短型gelonin（G-N3、G-C5、G-N3C5）. CD谱和荧光谱表明,完整型gelonin（G-O）与截短型gelonin的分子构象有明显的差异.它们的构象变化与类DNase活性和抑制肿瘤细胞生长的能力均为G-O≥ G-N3＞G C5＞G-N3C5. 结果再一次证明了具有α+β型结构蛋白,gelonin的构象与生物活性的一致性.     

大肠杆菌glgC基因的定点突变及功能分析

摘要: 利用PCR从Escherichia coli JM109基因组中扩增到全长为1 296 bp的glgC基因编码区, 通过PCR重组方法进行点突变, 获得氨基酸突变的3个突变体, 分别是Pro295Ser(Val121Ala, Met151Ile和Val334Asp)、Gly336 Asp单点突变和Pro295Ser/Gly336Asp(Lys109Arg), 其基因分别命名为295⁺³、336和295/ 336⁺¹。将突变和未突变的基因分别克隆到pET32-a, 构建重组质粒pET-glgC、pET-295⁺³、pET-336和pET-295/ 336+1, 在文中分别简称为a、b、c和d。转化大肠杆菌BL21(DE3), 在1 mmol/L IPTG 诱导下表达。SDS- PAGE 电泳分析显示, 在约67 kDa 处有1条明显与预期大小一致的蛋白质, 表明目的基因已得到融合表达。上述转化子的碘染和糖原含量测定结果, 第336位的Gly变成Asp后, 宿主菌的糖原含量提高; Pro295Ser/Gly336Asp(Lys109Arg)的突变导致宿主菌的糖原含量与Gly336Asp突变体相近, 表明在336突变基因的基础上增加Pro295Ser的突变没有进一步加大宿主菌中AGPase酶的反馈抑制效应的降低。已有的结果显示, Pro295Ser可以降低AGPase酶的反馈抑制效应活性, 而实验中295⁺³突变基因转入宿主菌后细胞糖原含量明显降低, 推测这个结果可能是295⁺³中的Val334Asp的突变造成, 而334位的氨基酸可能是AGPase功能域中的一个重要位点。     

Ydj1p二聚体中β14~β15与domain-III分离的拉伸分子动力学模拟研究

分子伴侣Hsp40是一种以二聚体的形式调控非天然多肽折叠的热激蛋白。本文通过拉伸分子动力学研究了酵母Hsp40家族成员Ydj1p二聚体中β14-β15与domain-Ⅲ的分离过程,深入探讨了影响Ydj1p二聚体稳定性的重要残基和相互作用力。研究表明,残基Thr366、Asp368、Cys370、Leu372和Phe375在Ydj1P二聚体的形成过程中发挥着重要的作用。其中,β14-β15中的残基Thr366和Asp368分别通过与domain-Ⅲ内的残基Asp291、Trp292和Trp292、Lys294之间形成的氢键,Asp368通过与domain-Ⅲ内的残基Lys314形成盐桥,Cys370、Leu372和Phe375则是通过与domain-Ⅲ形成疏水作用力来稳定Ydj1p二聚体结构。     

Correlation between absorption maxima and thermal isomerization rates in bacteriorhodopsin

The reported rates of thermal 13-cis to all-trans isomerization of the protonated Schiff base of retinal (PSBR) in solution and in bacteriorhodopsin (BR) are shown to be correlated with the red shift in the absorption maximum of the chromophore, though the linear fit is different for BR and for a model PSBR in solution. Because the red shift in the absorption has been previously shown to be correlated with π-electron delocalization in the chromophore, this suggests that the thermal isomerization rate is largely regulated by the amount of double bond character in the chromophore. Because the linear fit of isomerization rates with absorption maxima is different for BR and the model PSBR, specific interactions of the protein with the chromophore must also be a factor in determining thermal isomerization rates in BR. A model of the later steps in the photocycle of BR is presented in which the 13-cis to all-trans thermal isomerization occurs during the O intermediate.     

Structures of aspartic acid-96 in the L and N intermediates of bacteriorhodopsin: analysis by Fourier transform infrared spectroscopy.

The light-induced difference Fourier transform infrared spectrum between the L or N intermediate minus light-adapted bacteriorhodopsin (BR) was measured in order to examine the protonated states and the changes in the interactions of carboxylic acids of Asp-96 and Asp-115 in these intermediates. Vibrational bands due to the protonated and unprotonated carboxylic acid were identified by isotope shift and band depletion upon substitution of Asp-96 or -115 by asparagine. While the signal due to the deprotonation of Asp-96 was clearly observed in the N intermediate, this residue remained protonated in L. Asp-115 was partially deprotonated in L. The C = O stretching vibration of protonated Asp-96 of L showed almost no shift upon 2H2O substitution, in contrast to the corresponding band of Asp-96 or Asp-115 of BR, which shifted by 9-12 cm-1 under the same conditions. In the model system of acetic acid in organic solvents, such an absence of the shift of the C = O stretching vibration of the protonated carboxylic acid upon 2H2O substitution was seen only when the O-H of acetic acid is hydrogen-bonded. The non-hydrogen-bonded monomer showed the 2H2O-dependent shift. Thus, the O-H bond of Asp-96 enters into hydrogen bonding upon conversion of BR to L. Its increased hydrogen bonding in L is consistent with the observed downshift of the O-H stretching vibration of the carboxylic acid of Asp-96.     

Titration of aspartate-85 in bacteriorhodopsin: what it says about chromophore isomerization and proton release.

Titration of Asp-85, the proton acceptor and part of the counterion in bacteriorhodopsin, over a wide pH range (2-11) leads us to the following conclusions: 1) Asp-85 has a complex titration curve with two values of pKa; in addition to a main transition with pKa = 2.6 it shows a second inflection point at high pH (pKa = 9.7 in 150-mM KCl). This complex titration behavior of Asp-85 is explained by interaction of Asp-85 with an ionizable residue X'. As follows from the fit of the titration curve of Asp-85, deprotonation of X' increases the proton affinity of Asp-85 by shifting its pKa from 2.6 to 7.5. Conversely, protonation of Asp-85 decreases the pKa of X' by 4.9 units, from 9.7 to 4.8. The interaction between Asp-85 and X' has important implications for the mechanism of proton transfer. In the photocycle after the formation of M intermediate (and protonation of Asp-85) the group X' should release a proton. This deprotonated state of X' would stabilize the protonated state of Asp-85.2) Thermal isomerization of the chromophore (dark adaptation) occurs on transient protonation of Asp-85 and formation of the blue membrane. The latter conclusion is based on the observation that the rate constant of dark adaptation is directly proportional to the fraction of blue membrane (in which Asp-85 is protonated) between pH 2 and 11. The rate constant of isomerization is at least 10(4) times faster in the blue membrane than in the purple membrane. The protonated state of Asp-85 probably is important for the catalysis not only of all-trans <=> 13-cis thermal isomerization during dark adaptation but also of the reisomerization of the chromophore from 13-cis to all-trans configuration during N-->O-->bR transition in the photocycle. This would explain why Asp-85 stays protonated in the N and O intermediates.     

His-75 in Proteorhodopsin, a Novel Component in Light-driven Proton Translocation by Primary Pumps

Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near the photoactive site of the protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements of light-induced charge movements reveal participation of His-75 in the proton translocation mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the structure of the photoactive site and resulted in significantly shifted visible absorption spectra. In contrast, His-75 substitution with a positively charged Arg did not shift the visible absorption spectrum of PR. The mutation to Arg also blocks the light-induced proton transfer from the Schiff base to its counterion Asp-97 during the photocycle and the acid-induced protonation of Asp-97 in the dark state of the protein. Isotope labeling of histidine revealed that His-75 undergoes deprotonation during the photocycle in the proton-pumping (high pH) form of PR, a reaction further supported by results from H75E. Finally, all His-75 mutations greatly affect charge movements within the PR and shift its pH dependence to acidic values. A model of the proteorhodopsin proton transport process is proposed as follows: (i) in the dark state His-75 is positively charged (protonated) over a wide pH range and interacts directly with the Schiff base counterion Asp-97; and (ii) photoisomerization-induced transfer of the Schiff base proton to the Asp-97 counterion disrupts its interaction with His-75 and triggers a histidine deprotonation.A variety of unicellular microorganisms contain primary proton pumps that convert solar energy into a transmembrane electrochemical proton gradient, which is subsequently used by membrane ATP synthases to generate chemical energy. Well known examples of such pumps are the haloarchaeal rhodopsins, photoactive, seven-helix membrane proteins, which include the well studied proton pump bacteriorhodopsin (BR)⁴ from Halobacterium salinarum and BR homologs in other haloarchaea. Recently, a much larger new family of light-driven proton pumps, the proteorhodopsins (PRs), was identified in marine proteobacteria throughout the oceans (1–3). Despite the diverse properties of PRs, including different visible absorption maxima and photocycle rates (4–6), they all share with BR several key conserved residues as well as an all-trans-retinylidene chromophore in their unphotolyzed state, which is covalently bound to transmembrane helix G via a protonated Schiff base linkage.Many of the molecular events that occur in PRs following light activation are similar to those of BR, including an initial ultrafast all-trans→13-cis-retinal isomerization, which triggers a sequence of protein conformational changes, including several intramolecular proton transfer reactions. The two key carboxylate groups involved in proton pumping in helix C of BR are conserved in PRs, and in the first found and most commonly studied PR, the Monterey Bay variant eBAC31A08, also known as green-absorbing proteorhodopsin (GPR), the helix C residues Asp-97 and Glu-108 undergo protonation changes during the photocycle similar to those of the homologous carboxylate residues in BR. Initial FTIR studies on GPR identified the role of Asp-97 as the Schiff base counterion and proton acceptor during Schiff base deprotonation and concomitant M formation and Glu-108 as the proton donor that reprotonates the Schiff base during N formation (7, 8). Studies of other variants indicate these roles of the two carboxylic acid residues are general in the proteorhodopsin family.⁵One major difference between BR and the PRs is the presence of a highly conserved histidine residue at position 75, near the middle of transmembrane helix B in the latter pigments. The His-75 homolog is not present in BR nor thus far found in other microbial rhodopsins (9). The proximity of His-75 to the protein active site and specifically to the Schiff base counterion Asp-97 inferred from the x-ray crystal structure of BR suggests its involvement in spectral tuning of the visible absorption (10) and potentially PR photochemical reactions. Because the pK_a of histidine in solution is close to neutral pH (11), its imidazole group often plays a major role in intramolecular proton transfers in enzymes, including NADPH oxidase (12), alcohol dehydrogenase (13), carbonic anhydrase II (14), and serine proteases (15).In this study we have used a combination of time-resolved FTIR difference spectroscopy, visible absorption spectroscopy, isotope labeling, kinetic charge displacement measurements, and site-directed mutagenesis to study the role of His-75 in GPR. We report evidence that protonated His-75 interacts directly with Asp-97 in the unphotolyzed protein and during the photocycle undergoes a deprotonation in response to the protonation of Asp-97.     

FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)). The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M). The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH. In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.     

Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin

Bacteriorhodopsin (BR) with the single-site substitutions Arg-82----Gln (R82Q), Asp-85----Asn (D85N), and Asp-96----Asn (D96N) is studied with time-resolved absorption spectroscopy in the time regime from nanoseconds to seconds. Time-resolved spectra are analyzed globally by using multiexponential fitting of the data at multiple wavelengths and times. The photocycle kinetics for BR purified from each mutant are determined for micellar solutions in two detergents, nonyl glucoside and CHAPSO, and are compared to results from studies on delipidated BR (d-BR) in the same detergents. D85N has a red-shifted ground-state absorption spectrum, and the formation of an M intermediate is not observed. R82Q undergoes a pH-dependent transition between a purple and a blue form with different pKa values in the two detergents. The blue form has a photocycle resembling that for D85N, while the purple form of R82Q forms an M intermediate that decays more rapidly than in d-BR. The purple form of R82Q does not light-adapt to the same extent as d-BR, and the spectral changes in the photocycle suggest that the light-adapted purple form of R82Q contains all-trans- and 13-cis-retinal in approximately equal proportions. These results are consistent with the suggestions of others for the roles of Arg-82 and Asp-85 in the photocycle of BR, but results for D96N suggest a more complex role for Asp-96 than previously suggested. In nonyl glucoside, the apparent decay of the M-intermediate is slower in D96N than in d-BR, and the M decay shows biphasic kinetics. However, the role of Asp-96 is not limited to the later steps of the photocycle. In D96N, the decay of the KL intermediate is accelerated, and the rise of the M intermediate has an additional slow phase not observed in the kinetics of d-BR. The results suggest that Asp-96 may play a role in regulating the structure of BR and how it changes during the photocycle.     

Isoaspartate formation and neurodegeneration in Alzheimer's disease

We reviewed here that protein isomerization is enhanced in amyloid-beta peptides (Abeta) and paired helical filaments (PHFs) purified from Alzheimer's disease (AD) brains. Biochemical analyses revealed that Abeta purified from senile plaques and vascular amyloid are isomerized at Asp-1 and Asp-7. A specific antibody recognizing isoAsp-23 of Abeta further suggested the isomerization of Abeta at Asp-23 in vascular amyloid as well as in the core of senile plaques. Biochemical analyses of purified PHFs also revealed that heterogeneous molecular weight tau contains L-isoaspartate at Asp-193, Asn-381, and Asp-387, indicating a modification, other than phosphorylation, that differentiates between normal tau and PHF tau. Since protein isomerization as L-isoaspartate causes structural changes and functional inactivation, or enhances the aggregation process, this modification is proposed as one of the progression factors in AD. Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of isomerized proteins containing L-isoaspartate. We show here that PIMT is upregulated in neurodegenerative neurons and colocalizes in neurofibrillary tangles (NFTs) in AD. Taken together with the enhanced protein isomerization in AD brains, it is implicated that the upregulated PIMT may associate with increased protein isomerization in AD. We also reviewed studies on PIMT-deficient mice that confirmed that PIMT plays a physiological role in the repair of isomerized proteins containing L-isoaspartate. The knockout study also suggested that the brain of PIMT-deficient mice manifested neurodegenerative changes concomitant with accumulation of L-isoaspartate. We discuss the pathological implications of protein isomerization in the neurodegeneration found in model mice and AD.     

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)     

Light- and dark-adapted bacteriorhodopsin, a time-resolved neutron diffraction study

Recently, neutron diffraction experiments have revealed well-resolved and reversible changes in the protein conformation of bacteriorhodopsin (BR) between the light-adapted ground state and the M-intermediate of the proton pumping photocycle (Dencher, Dresselhaus, Zaccai and Büldt (1989) Proc. Natl. Acad. Sci. USA 86, 7876-7879). These changes are triggered by the light-induced isomerization of the chromophore retinal from the all-trans to the 13-cis configuration. Dark-adapted purple membranes contain a mixture of two pigment species with either the all-trans- or 13-cis-retinal isomer as chromophore. Employing a time-resolved neutron diffraction technique, no changes in protein conformation in the resolution regime of up to 7 A are observed during the transition between the two ground-state species 13-cis-BR and all-trans-BR. This is in line with the fact that the conversion of all-trans BR to 13-cis-BR involves an additional isomerization about the C15 = N Schiff's base bond, which in contrast to M formation minimizes retinal displacement and keeps the Schiff's base in the original protein environment. Furthermore, there is no indication for large-scale redistribution of water molecules in the purple membrane during light-dark adaptation.     

Structural changes of water in the Schiff base region of bacteriorhodopsin: proposal of a hydration switch model

In a light-driven proton-pump protein, bacteriorhodopsin (BR), three water molecules participate in a pentagonal cluster that stabilizes an electric quadrupole buried inside the protein. In low-temperature Fourier transform infrared (FTIR) K minus BR spectra, the frequencies of water bands suggest extremely strong hydrogen bonding conditions in BR. The three observed water O-D stretches, at 2323, 2292, and 2171 cm(-1), are probably associated with water that interacts with the negative charges in the Schiff base region. Retinal isomerization weakens these hydrogen bonds in the K intermediate, but not in the later intermediates such as L, M, and N. In these states, spectral changes of water bands appeared only in the >2500 cm(-1) region, which correspond to weak hydrogen bonds. This observation suggests that after the K state the water molecules in the Schiff base region find a hydrogen bonding acceptor. We propose here a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have increased the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85. The present results also suggest that the deprotonated Asp96 in the N intermediate is stabilized in a manner different from that of Asp85 in BR.