Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm.     

Self-assembled monolayer of light-harvesting core complexes from photosynthetic bacteria on a gold electrode modified with alkanethiols

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodoseudomonas palustris were self-assembled on a gold electrode modified with self-assembled monolayers (SAMs) of the alkanethiols NH2(CH2)nSH, n = 2, 6, 8, 11; HOOC(CH2)7SH; and CH3(CH2)7SH, respectively. Adsorption of the LH1-RC complexes on the SAMs depended on the terminating group of the alkanethiols, where the adsoption increased in the following order for the terminating groups: amino groups > carboxylic acid groups > methyl groups. Further, the adsorption on a gold electrode modified with SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, depended on the methylene chain length, where the adsorption increased with increasing the methylene chain length. The presence of the well-known light-harvesting and reaction center peaks of the near infrared (NIR) absorption spectra of the LH1-RC complexes indicated that these complexes were only fully stable on the SAM gold electrodes modified with the amino group. In the case of modification with the carboxyl group, the complexes were partially stable, while in the presence of the terminal methyl group the complexes were extensively denatured. An efficient photocurrent response of these complexes on the SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, was observed upon illumination at 880 nm. The photocurrent depended on the methylene chain length (n), where the maximum photocurrent response was observed at n = 6, which corresponds to a distance between the amino terminal group in NH2(CH2)6SH and the gold surface of 1.0 nm.     

Orientated binding of photosynthetic reaction centers on gold using Ni-NTA self-assembled monolayers

Coupling of photosynthetic reaction centers (RCs) with inorganic surfaces is attractive for the identification of the mechanisms of interprotein electron transfer (ET) and for possible applications in construction of photo- and chemosensors. Here we show that RCs from Rhodobacter sphaeroides can be immobilized on gold surfaces with the RC primary donor looking towards the substrate by using a genetically engineered poly-histidine tag (His₇) at the C-terminal end of the M-subunit and a Ni---NTA terminated self-assembled monolayer (SAM). In the presence of an electron acceptor, ubiquinone-10, illumination of this RC electrode generates a cathodic photocurrent. The action spectrum of the photocurrent coincides with the absorption spectrum of RC and the photocurrent decreases in response to the herbicide, atrazine, confirming that the RC is the primary source of the photoresponse. Disruption of the Ni---NTA---RC bond by imidazole leads to about 80% reduction of the photocurrent indicating that most of the photoactive protein is specifically bound to the electrode through the linker.     

Reconstitution of photosynthetic reaction centers and core antenna-reaction center complexes in liposomes and their thermal stability

Photosynthetic reaction centers (RCs) and their core light-harvesting complexes (LH1-RCs), purified from a thermophile, Thermochromatium (T.) tepidum, and a mesophile, Allochromatium (A.) vinosum, were reconstituted into liposomes. The RC and the LH1-RC in the reconstituted liposomes were found intact from the absorption spectra at about 4 and 40 degrees C respectively. The thermal stability of the RCs of T. tepidum in the liposome was dependent on whether they were surrounded directly by lipids or by the core light-harvesting complexes. The results show that the RC of T. tepidum gains its thermostability through interactions with the LH1. These results are consistent with the result that the thermal stability of the LH1 in T. tepidum is similar in both the reconstituted LH1-RC liposome and ICM. This is clearly different from the mesophilic bacterium, A. vinosum. The thermal stability of RC was also affected by its subunit constitution: the RC containing a cytochrome subunit was more thermostable than the cytochrome-detached RC. This suggests that the cytochrome subunit might play a role in protecting the special pair pigments from denaturation. The thermal denaturation showed a second-order reaction dependence on time. The interaction of the pigments with proteins and/or lipids might be the cause of the second-order reaction profile.     

Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer

We present a novel infrared method to investigate the functionality of a protein monolayer tethered to a metal substrate. The approach employs Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS), which renders high surface sensitivity by enhancing the signal of the adsorbed protein by up to approximately 2 orders of magnitude. We demonstrate that the electrochemically induced absorption changes of a cytochrome c monolayer can be observed with excellent signal-to-noise ratio when the protein is adhered to a modified gold surface. To probe membrane proteins, a concept is introduced for the oriented incorporation into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent is immobilized on a chemically modified gold surface via the affinity of its histidine (His)-tag to a nickel-chelating nitro-triacetic acid (NTA) surface. The protein monolayer is reconstituted into the lipid environment by detergent removal. Changing the orientation of the protein with respect to the metal surface is achieved by inserting the His-tag on either side of the membrane protein surface. Orientational control is mandatory for experiments in which electrons are injected from the electrode into the protein. The presented methodology opens new avenues to study the mechanism of the biomedically relevant class of electron and voltage-gated proteins on the atomic level.     

Circular symmetry of the light-harvesting 1 complex from Rhodospirillum rubrum is not perturbed by interaction with the reaction center

The effect of the interaction of the reaction center (RC) upon the geometrical arrangement of the bacteriochlorophyll a (BChla) pigments in the light-harvesting 1 complex (LH1) from Rhodospirillum rubrum has been examined using single molecule spectroscopy. Fluorescence excitation spectra at 1.8 K obtained from single detergent-solubilized as well as single membrane-reconstituted LH1-RC complexes showed predominantly (>70%) a single broad absorption maximum at 880-900 nm corresponding to the Q(y) transition of the LH1 complex. This absorption band was independent of the polarization direction of the excitation light. The remaining complexes showed two mutually orthogonal absorption bands in the same wavelength region with moderate splittings in the range of DeltaE = 30-85 cm(-1). Our observations are in agreement with simulated spectra of an array of 32 strongly coupled BChla dipoles arranged in perfect circular symmetry possessing only a diagonal disorder of 相似文献   

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q(y) transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (alpha and beta) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q(y) transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 A.     

Use of new strains of Rhodobacter sphaeroides and a modified simple culture medium to increase yield and facilitate purification of the reaction centre

A new gene expression system was developed in Rhodobacter sphaeroides, replacing a pRK415-based system used previously. The broad host-range IPTG-inducible plasmid pIND4 was used to create the plasmid pIND4-RC1 for expression of the puhA and pufQBALMX genes, encoding the reaction centre (RC) and light-harvesting complex 1 (LH1) proteins. The strain R. sphaeroides ΔRCLH was used to make a knockout of the rshI restriction endonuclease gene, enabling electroporation of DNA into the bacterium; a subsequent knockout of ppsR was made, creating the strain R. sphaeroides RCx lacking this oxygen-sensing repressor of the photosynthesis gene cluster. Using pIND4-RC1, LH1 levels were increased by a factor of about 8 over pRS1 per cell in cultures grown semi-aerobically. In addition, the ppsR knockout allowed for photosynthetic pigment–protein complex synthesis in the presence of high concentrations of molecular oxygen; here, LH1 levels per cell increased by 20 % when grown under high aeration conditions. A new medium (called RLB) is the E. coli medium LB supplemented with MgCl₂ and CaCl₂, which was found to increase growth rates and final cell culture densities, with an increase of 30 % of LH1 per cell detected in R. sphaeroides RCx(pIND4-RC1) grown in RLB versus LB medium. Furthermore, cell density was about three times greater in RLB compared to semi-aerobic conditions. The combination of all the modifications resulted in an increase of LH1 and RC per mL of culture volume by approximately 35-fold, and a decrease in the length of culture incubation time from about 5 days to ~36 h.     

Role of the N- and C-terminal regions of the PufX protein in the structural organization of the photosynthetic core complex of Rhodobacter sphaeroides.

The core complex of Rhodobacter sphaeroides is formed by the association of the light-harvesting antenna 1 (LH1) and the reaction center (RC). The PufX protein is essential for photosynthetic growth; it is located within the core in a 1 : 1 stoichiometry with the RC. PufX is required for a fast ubiquinol exchange between the Q(B) site of the RC and the Qo site of the cytochrome bc1 complex. In vivo the LH1-PufX-RC complex is assembled in a dimeric form, where PufX is involved as a structural organizer. We have modified the PufX protein at the N and the C-terminus with progressive deletions. The nine mutants obtained have been characterized for their ability for photosynthetic growth, the insertion of PufX in the core LH1-RC complex, the stability of the dimers and the kinetics of flash-induced reduction of cytochrome b561 of the cytochrome bc1 complex. Deletion of 18 residues at the N-terminus destabilizes the dimer in vitro without preventing photosynthetic growth. The dimer (or a stable dimer) does not seem to be a necessary requisite for the photosynthetic phenotype. Partial C-terminal deletions impede the insertion of PufX, while the complete absence of the C-terminus leads to the insertion of a PufX protein composed of only its first 53 residues and does not affect the photosynthetic growth of the bacterium. Overall, the results point to a complex role of the N and C domains in the structural organization of the core complex; the N-terminus is suggested to be responsible mainly for dimerization, while the C-terminus is thought to be involved mainly in PufX assembly.     

Spirilloxanthin incorporation into the LH2 and LH1-RC pigment-protein complexes from a purple sulfur bacterium <Emphasis Type="Italic">Allochromatium minutissimum</Emphasis>

Incorporation of spirilloxanthin into carotenoidless LH2 and LH1-RC complexes from a purple sulfur bacterium Allochromatium (Alc.) minutissimum was studied. Carotenoidless cells of Alc. minutissimum were obtained using diphenylamine, a carotenoid biosynthesis inhibitor. In the course of incorporation of the carotenoid mixture, the composition of which corresponded to that of Alc. minutissimum control photosynthetic membranes, no selective incorporation of spirilloxanthin into the LH1-RC complex was detected. It is assumed that in vivo carotenoids are not incorporated into the LH2 and LH1-RC complexes from a common pool. Pure spirilloxanthin destroys both the LH2 and LH1-RC complexes. Within the concentration range of spirilloxanthin in the incorporated mixture from 27% to 52%, it was found to be incorporated into the LH2 and LH1-RC complexes with the efficiency of 13% and 33%, respectively. The possible existence of different sites of assembly for the LH2 and LH1-RC complexes is discussed, as well as of two fractions of LH2 complexes, in one of which rhodopin may be integrated, and in the other (minor) one, spirilloxanthin.     

新载体pET-DB对带有His-tag的仓鼠二氢叶酸还原酶的高效表达与纯化

报道了带有His-tag的仓鼠二氢叶酸还原酶基因的克隆和在DB序列增强下T7启动子调控该基因在大肠杆菌中的可溶性高效表达,SDS-PAGE分析表明,带有His-tag的仓鼠二氢叶酸还原酶的含量可占大肠杆菌细胞总蛋白质含量的46%.该酶的纯化可用常规的金属络合树脂一步纯化至SDS-PAGE一条带,经凝血酶切去His-tag的仓鼠二氢叶酸还原酶与用等电聚焦法获得的无His-tag的酶有相同的酶活性.     

Calcium ions are required for the enhanced thermal stability of the light-harvesting-reaction center core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

Thermochromatium tepidum is a thermophilic purple sulfur photosynthetic bacterium collected from the Mammoth Hot Springs, Yellowstone National Park. A previous study showed that the light-harvesting-reaction center core complex (LH1-RC) purified from this bacterium is highly stable at room temperature (Suzuki, H., Hirano, Y., Kimura, Y., Takaichi, S., Kobayashi, M., Miki, K., and Wang, Z.-Y. (2007) Biochim. Biophys. Acta 1767, 1057-1063). In this work, we demonstrate that thermal stability of the Tch. tepidum LH1-RC is much higher than that of its mesophilic counterparts, and the enhanced thermal stability requires Ca2+ as a cofactor. Removal of the Ca2+ from Tch. tepidum LH1-RC resulted in a complex with the same degree of thermal stability as that of the LH1-RCs purified from mesophilic bacteria. The enhanced thermal stability can be restored by addition of Ca2+ to the Ca2+-depleted LH1-RC, and this process is fully reversible. Interchange of the thermal stability between the two forms is accompanied by a shift of the LH1 Qy transition between 915 nm for the native and 880 nm for the Ca2+-depleted LH1-RC. Differential scanning calorimetry measurements reveal that degradation temperature of the native LH1-RC is 15 degrees C higher and the enthalpy change is about 28% larger than the Ca2+-depleted LH1-RC. Substitution of the Ca2+ with other metal cations caused a decrease in thermal stability of an extent depending on the properties of the cations. These results indicate that Ca2+ ions play a dual role in stabilizing the structure of the pigment-membrane protein complex and in altering its spectroscopic properties, and hence provide insight into the adaptive strategy of this photosynthetic organism to survive in extreme environments using natural resources.     

Construction and structural analysis of tethered lipid bilayer containing photosynthetic antenna proteins for functional analysis

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAP), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.     

Self-assembly of semiconductor quantum-dots on electrodes for photoelectrochemical biosensing.

CdS nanoparticles linked through a duplex DNA to a Au electrode do not lead to a noticeable photocurrent upon their illumination in the presence of triethanolamine, TEOA, 20 mM, pH = 7.2. The intercalation of doxorubicin into the duplex DNA stimulates, however, the generation of a photocurrent. This is attributed to the trapping of photoexcited conduction-band electrons by the intercalator units that facilitates, by a hopping mechanism, the electron transport to the electrode. The oxidation of TEOA by valence band holes allows the formation of a steady state photocurrent. This basic phenomenon is used to probe the operation of a DNA-based machine through the assembly of CdS nanoparticles on a Au electrode. The machine includes a nucleic acid "track", (1), that binds a primer, (2), through hybridization to a predefined domain. In the presence of polymerase, the nucleotide mixture, dNTPs, and the nicking enzyme, the autonomous replication, nicking and displacement of the "waste product", (3), are activated. The "waste product" bridges the (5)-functionalized CdS nanoparticles and the nucleic acid (4)-functionalized Au electrode, resulting in the assembly of the nanoparticles on the electrode. The intercalation of doxorubicin into the DNA-CdS nanostructures results in the generation of photocurrents upon illumination in the presence of TEOA, pH = 7.2. The photocurrents are controlled by the time intervals used to operate the DNA machine.     

Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme,Sphingobium fuliginis ATCC 27551

The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.     

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q_y transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (α and β) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q_y transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 Å.     

Engineering, purification and applications of His-tagged recombinant antibody fragments with specificity for the major birch pollen allergen, bet v1

Type I allergy, an immunodisorder affecting almost 20% of the population worldwide, is based on the production of IgE antibodies against per se harmless allergens. We report the expression of hexahistidine-tagged antibody fragments (Fabs) with specificity for Bet v1, the major birch pollen allergen, in Escherichia coli. The cDNA coding for the heavy chain fragment of a mouse monoclonal anti-Bet v1 antibody, Bip 1, was engineered by PCR to contain a hexahistidine-encoding 3' end. The modified Bip1 heavy chain cDNA was co-expressed in E. coli XL-1 Blue with the Bip 1 light chain cDNA using the combinatorial plasmid pComb3H. His-tagged recombinant (r) Bip 1 Fabs were isolated by nickel affinity chromatography and rBip 1 Fabs without His-tag were purified via affinity to rBet v1. rBip 1 Fabs with and without His-tag bound specifically to rBet v1 and, like Bet v1 -specific human serum IgE and rabbit-anti rBet v1 antibodies, cross-reacted with Bet v1-related allergens in other plant-species (alder, oak, hazelnut). We demonstrate the usefulness of His-tagged rBip 1 Fabs (1) for the identification of pollen samples containing Bet v 1 by particle blotting, (2) forthe detection of Bet v1-specific IgE antibodies in human serum samples by sandwich ELISA and (3) for the quantification of Bet v1 in solution. Based on these examples we suggest to use rBip 1 Fabs for the detection of Bet v1 and Bet v1-related allergens in natural allergen sources for allergy prevention, as well as for the standardization of natural allergen extracts produced for diagnosis and immunotherapy of birch pollen allergy.     

Construction of efficient bioelectrochemical devices: Improved electricity production from cyanobacteria (Leptolyngbia sp.) based on π-conjugated conducting polymer/gold nanoparticle composite interfaces

In this study, gold electrodes (GE) were coated with conducting polymers to obtain a high photocurrent using cyanobacteria from a novel bioelectrochemical fuel cell. For this purpose, 4-(4H-ditiheno[3,2-b:2',3'-d]pyrol-4-yl) aniline and 5-(4H-dithieno[3,2-b:2',3'-d]pyrol-4-yl) napthtalane-1-amine monomers were coated on GE by performing an electropolymerization process. After that, gold nanoparticles (AuNP) were specifically modified by 2-mercaptoethane sulfonic acid and p-aminothiophenol to attach to the electrode surface. The conducting polymers GE coat was modified with functionalized AuNP using a cross-linker. The resulting electrode structures were characterized by cyclic voltammetry and chronoamperometry under on-off illumination using a fiber optic light source. Cyanobacteria Leptolyngbia sp. was added to the GE/conducting polymer/AuNP electrode surface and stabilized by using a cellulose membrane. During the illumination, water was oxidized by the photosynthesis, and oxygen was released. The released oxygen was electrocatalytically reduced at the cathode surface and a 25 nA/cm ² photocurrent was observed in GE/ Leptolyngbia sp. After the electrode modifications, a significant improvement in the photocurrent up to 630 nA/cm ² was achieved.     

Differentiating the orientations of photosynthetic reaction centers on Au electrodes linked by different bifunctional reagents

The photosynthetic reaction center (RC) composite film was fabricated by self-assembled monolayers (SAMs) on the Au electrode with two different bifunctional reagents, 4-aminothiophenol (ATP) and 2-mercaptoethylamine (MEA), respectively. The square wave voltametry (SWV), bulk electrolysis and photocurrent test were employed for characterizing the composite film. The dramatic different electrochemical characteristics were observed for the two types of films, which strongly suggested an orientational difference for RC arising from the structural difference between the two bifunctional reagents. For RC-MEA film, three redox peaks which implying electron transfer (ET) between the primary donor (P) and the bacteriopheophytin (Bphe) were observed. While for RC-ATP film, two redox peaks implying ET between the nonheme iron and the primary quinone (Q(A)) were observed. The ET behavior driven by electric field also supported the result that the RC could be linked to the electrode at different sites. The site-specific immobilization approach reported here supplies a method to differentiate the protein orientation.     

Targeted protein functionalization using His-tags

With the impressive growth in gene sequence data that has become available, recombinant proteins represent an increasingly vast source of molecular components, with unique functional and structural properties, for use in biotechnological applications and devices. To facilitate the use, manipulation, and integration of such molecules into devices, a controllable method for their chemical modification was developed. In this approach, a trifunctional labeling reagent first recognizes and binds a His-tag on the target protein's surface. After binding, a photoreactive group on the trifunctional molecule is triggered to create a covalent linkage between the reagent and the target protein. The third moiety on the labeling reagent can be varied to bring unique chemical functionality to the target protein. This approach provides: (1) specificity in that only His-tagged targets are modified, (2) regio-specific control in that the target is modified proximal to the His-tag, the position of which can be varied, and (3) stoichiometric control in that the number modifications is limited by the binding capacity of the His-tag. Two such labeling reagents were designed, synthesized, and used to modify both N- and C-terminally His-tagged versions of the enzyme murine dihydrofolate reductase (mDHFR). The first reagent biotinylated the enzyme,while the second served to attach an oligonucleotide to yield a protein-DNA conjugate. In all cases, modification in this manner brings new functionality to the protein while leaving the enzymatic activity intact. The protein-DNA conjugate was used to specifically immobilize the active enzyme through DNA hybridization onto polystyrene microspheres, a step toward creating a functional protein microarray.