Preparation and characterization of a chemically modified plastocyanin.

The reaction of plastocyanin with tetranitromethane results in the nitration of only one of the three tyrosyl residues present in the protein. The modification does not affect the blue copper chromophore as both the characteristic visible spectrum of the chromophore and the redox potential of the protein are unchanged. Photochemical assays show that the modified plastocyanin is fully active in the reduction of photooxidized P700 and in the photooxidation of cytochrome f. The pK of the nitro-tyrosyl residue is about 7.3 indicating that the modified residue may be located in a negatively charged environment. Examination of the recently published X-ray structure of poplar plastocyanin suggests that Tyr-80 would be a likely candidate for the site of modification.     

Functional characterization of the evolutionarily divergent fern plastocyanin.

Plastocyanin (Pc) is a soluble copper protein that transfers electrons from cytochrome b(6)f to photosystem I (PSI), two protein complexes that are localized in the thylakoid membranes in chloroplasts. The surface electrostatic potential distribution of Pc plays a key role in complex formation with the membrane-bound partners. It is practically identical for Pcs from plants and green algae, but is quite different for Pc from ferns. Here we report on a laser flash kinetic analysis of PSI reduction by Pc from various eukaryotic and prokaryotic organisms. The reaction of fern Pc with fern PSI fits a two-step kinetic model, consisting of complex formation and electron transfer, whereas other plant systems exhibit a mechanism that requires an additional intracomplex rearrangement step. The fern Pc interacts inefficiently with spinach PSI, showing no detectable complex formation. This can be explained by assuming that the unusual surface charge distribution of fern Pc impairs the interaction. Fern PSI behaves in a similar way as spinach PSI in reaction with other Pcs. The reactivity of fern Pc towards several soluble c-type cytochromes, including cytochrome f, has been analysed by flavin-photosensitized laser flash photolysis, demonstrating that the specific surface motifs for the interaction with cytochrome f are conserved in fern Pc.     

The fossil hornwort described from Dominican amber is an angiosperm flower

Hornworts (Anthocerotophyta) are a main lineage of land plants but they are exceedingly rare as fossils. The only fossil hornwort described from amber has been interpreted as the best preserved fossil of this group. Reinvestigation of this fossil revealed that this Miocene amber inclusion represents a poorly preserved flower that shows some features of the Caesalpinioideae subfamily of the Fabaceae.     

Spectroscopic characterization of Phaseolus vulgaris leucoagglutinin

Phaseolus vulgaris leucoagglutinin is a homotetrameric legume lectin possessing the canonical dimeric structure common to other legume lectins. In order to gain insight into the stability of the protein in an acidic environment, it was characterized by CD and fluorescence studies at pH 2.5. This was then compared with the native protein at physiological pH (7.2). The extinction coefficient of the native protein was calculated to be 3.58x10(4) from its UV absorption spectra. The far- and near-UV CD spectra of the protein at pH 2.5 showed very little difference even though the protein was found to exist as a dimer at pH 2.5. The fluorescence emission maxima of the protein upon excitation at 280 nm were found to shift only from 331 nm at pH 7.2 to 333 nm at pH 2.5. Based on the above observation it was concluded that the protein exhibits extreme pH stability especially in the acidic range. The secondary and tertiary structure of the protein is lost only when it is incubated for two days in 6 M GdnHCl at pH 2.5. At pH 7.2 it could be denatured in 6 M GdnHCl after one week of incubation.     

Spectroscopic characterization of a truncated hemoglobin from the nitrogen-fixing bacterium Herbaspirillum seropedicae

The Herbaspirillum seropedicae genome sequence encodes a truncated hemoglobin typical of group II (Hs-trHb1) members of this family. We show that His-tagged recombinant Hs-trHb1 is monomeric in solution, and its optical spectrum resembles those of previously reported globins. NMR analysis allowed us to assign heme substituents. All data suggest that Hs-trHb1 undergoes a transition from an aquomet form in the ferric state to a hexacoordinate low-spin form in the ferrous state. The close positions of Ser-E7, Lys-E10, Tyr-B10, and His-CD1 in the distal pocket place them as candidates for heme coordination and ligand regulation. Peroxide degradation kinetics suggests an easy access to the heme pocket, as the protein offered no protection against peroxide degradation when compared with free heme. The high solvent exposure of the heme may be due to the presence of a flexible loop in the access pocket, as suggested by a structural model obtained by using homologous globins as templates. The truncated hemoglobin described here has unique features among truncated hemoglobins and may function in the facilitation of O₂ transfer and scavenging, playing an important role in the nitrogen-fixation mechanism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification of an acidic plastocyanin from Microcystis aeruginosa

Plastocyanin and cytochrome c-553 are two functionally equivalent electron carriers in the photosynthetic chain of cyanobacteria. Microcystis aeruginosa, a unicellular cyanobacterium which grows well at a high pH (8.6) and which was not known to possess plastocyanin, has been studied for its ability to synthesize plastocyanin in culture media with and without Cu. In the absence of Cu, an acidic cytochrome c-553 alone was isolated. With the inclusion of 2 microM Cu, cytochrome c-553 synthesis was partially suppressed and an acidic plastocyanin was isolated. A newly developed procedure, using high concentrations of ammonium sulfate to fractionate water-soluble proteins on Sephacryl S-200 was successfully used to isolate and concentrate the plastocyanin, thus allowing it to be further purified to homogeneity. This protein has an isoelectric point of 4.8 which is similar to the pI value reported for other acidic plastocyanins from higher plants and green algae. Its N-terminal sequence of the first 15 amino acids has been determined; 9 of these amino acids are identical to those in the sequence of the basic plastocyanin from Anabaena variabilis.     

Spectroscopic characterization of metallothionein from the terrestrial snail, Helix pomatia.

The Cd-sequestering metallothionein (MT) isoform isolated from the midgut gland of Roman snails exposed to Cd supplements in the feed was characterized by compositional and spectroscopic analysis. The preparations contained nearly 5 mol of Cd, small amounts of Cu and about 1 mol of Zn per chain mass of 6620 Da, in numerical agreement with the apoprotein's measured capacity of firmly binding a maximum of 6 equivalents of Cd per molecule. As with other Cd-containing MTs the occurrence of a prominent Cd-mercaptide-specific shoulder at 250 nm in its absorption spectrum showed that Cd is complexed in tetrahedral symmetry by the cysteine residues of the protein, and the multiphasic ellipticity profile in the CD spectrum revealed that these complexes are joined to form one or more oligonuclear Cd-mercapto clusters. Both spectral features vanished with the removal of the metal but were reconstituted to maximum amplitudes by readdition of Cd to the metal-free apoprotein, provided precautions were taken to prevent air oxidation of the latter. Quantitative analysis of snail MT reconstituted with Cd established that the 18 cysteine side chains bind the metal in a 3-to-1 ratio; spectroscopic studies on fractionally restored forms demonstrated that the six Cd ions were bound to the apoprotein molecule in succession in two sets of three Cd ions each. Thus, one can infer from the observed stoichiometry and the coordinating preferences of Cd that this gastropod MT, like the Cd-bearing MTs of marine crustaceans, harboured the metal in two separate cyclically constructed Cd3Cys9 clusters. The snail clusters differed, however, from other MTs in their response to acidification. Their protolytic dissociation proceeded through two separate protonation steps with the manifestation of spectroscopically distinguishable intermediate forms. Thus, this snail isoform displays in its metal composition and its chemical and spectroscopic features both similarities and differences to other animal kingdom MTs. Its properties suggest that it serves an important role in the protection of the terrestrial gastropod from Cd.     

Spectroscopic characterization and structural modeling of prolamin from maize and pearl millet

Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize (Zea mays) and pearl millet (Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high -helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, ¹H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded -helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.     

Spectroscopic characterization of the artificial Siderophore pyridinochelin

Siderophores play a very important role in the uptake process of iron by bacteria. Due to the so-called active transport the uptake of siderophores by bacteria is very specific, which makes the use of siderophores as effective shuttles for antibiotics in the treatment of infections and other diseases caused by bacteria highly attractive. In order to further investigate the transport and incorporation of siderophores into the bacteria cells, distinct molecular probes are needed. Especially artificial siderophores, that show a specific intrinsic fluorescence, are highly attractive for such monitoring purposes. A promising candidate of such a fluorescent artificial siderophore is bis-2,3-dihydroxybenzoyl-2,6-dimethylamino-pyridine (pyridinochelin, PY). The fluorescence properties of PY were investigated in different solvents and in the presence of different metal ions. It was found that PY in its free form shows a complex fluorescence behavior. In methanol a clear dual fluorescence is observed. In aqueous solution intermolecular interactions with water molecules are determining the intrinsic fluorescence. Upon complexation with metal ions (Me3+ = Eu3+, Tb3+, Al3+, Fe3+) the fluorescence characteristics changed. The fluorescence quantum yield of PY decreased upon addition of Me3+--except for Al3+, which showed no fluorescence quenching. The fluorescence decay of PY loaded with metal ions showed a nicely mono-exponential fluorescence decay, which was in contrast to PY in the absence of metal ions. This drastic change in the fluorescence properties of PY upon metal ion complexation makes PY highly attractive as a fluorescence probe for the investigation of siderophore action and siderophore-mediated transport processes.     

Spectroscopic characterization of heat-induced nonnative beta-lactoglobulin monomers

Previous studies have shown that two altered monomeric species were formed in the early steps of thermal denaturation of bovine beta-lactoglobulin (beta-lg), the well-known Cys121-exposed intermediate (Mcys121), and a new, stable monomer with exposed nonnative Cys119 (Mcys119). In this study, circular dichroism and fluorescence spectroscopies were used to characterize the structural features of these molecules. The structural characteristics of MCys121 after heating and cooling cycles are similar to those of native beta-lg. In contrast, Mcys119 monomer exhibits some characteristics of the well-known molten-globule state. Combined with other published data, these results indicate that heating induces at least two molten globule-like states of beta-lg, a highly reactive Mcys121 that returns to native state after cooling, and a less-reactive Mcys119 that is trapped and stabilized in a molten globule-like state by nonnative disulfide bond.     

Spectroscopic and functional characterization of human beta-synuclein

To elucidate potential biological roles of human beta-synuclein, we studied its conformational changes under various conditions. The structural and functional properties of beta-synuclein were characterized using biochemical and bio-physical methods including: a functional assay, mass spectrometry, size exclusion chromatography, circular dichroism (CD), and fluorescence spectroscopy. The results showed beta-synuclein has a high proportion of random coil in solution.     

Spectroscopic characterization of poly(Glu-Ala)

Infrared linear dichroism and ultraviolet circular dichroism speetroscopy have been used to distinguish four conformational forms of the ionizable sequential polypeptide poly(Glu-Ala). Two of these conformations, the α helix and the β form, were observed for the unionized polypeptide in solution. The α helix appeared immediately upon neutralization of the side-chain carboxyl functions, whereas the β form was observed after the neutralized solution had been standing for several days. The β form was also observed for films cast from either high or low pH solutions. Ionization of the glutamyl residues resulted in a circular dichroism spectrum which has previously been observed for charged homopolymers and appears to result from an extended helical conformation. Further, heating either the α helical or the charged extended helix resulted in a transition to a disordered chain. These results are consistent with the results of conformational calculations presented elsewhere.     

Spectroscopic characterization of the Stentor photoreceptor.

1. On the basis of chromatographic and spectroscopic (absorption, fluorescence and its polarization, fluorescence lifetime, circular dichroism) characterization of the Stentor photoreceptor (stentorin) for photophobic response, the photoreceptor chromophore released from mild acid hydrolysis has been identified as hypericin. 2. The native chromophore is apparently linked to a protein (65 K) containing Lys and several hydrophobic residues, which is soluble in acetone and n-pentane. The peptide-linked stentorin (I) chromophore exhibits circular dichroism in the visible region due to the induced optical activity provided by the peptide. 3. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of a 38% fraction of the sucrose density centrifugation has resolved stentorin II proteins having molecular weights of 13 000, 16 000, 65 000 and 130 000. These proteins, as well as the acetone-soluble peptide, have been spectroscopically characterized with particular emphasis on their primary photoreactivity as the photophobic receptor of Stentor coeruleus. 4. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium. A strong dependence upon pH in the fluorescence spectra of both synthetic and native chromophores is also evident, showing a significant drop in the pKa of one or more hydroxyl groups in the excited state. A mechanism for the photophobic response, based on this lowering of the pKa as the primary photoprocess, has been discussed.     

Electron transfer from plastocyanin to photosystem I.

Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site-specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C-terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid-targeting domain that acts as a bacterial export signal. The identity of the purified plastocyanins was verified by matrix-assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin. We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.     

Spectroscopic characterization of streptavidin functionalized quantum dots

The spectroscopic properties of quantum dots can be strongly influenced by the conditions of their synthesis. In this work, we have characterized several spectroscopic properties of commercial, streptavidin functionalized quantum dots (QD525, lot 1005-0045, and QD585, lot 0905-0031, from Invitrogen). This is the first step in the development of calibration beads to be used in a generalizable quantification scheme of multiple fluorescent tags in flow cytometry or microscopy applications. We used light absorption, photoexcitation, and emission spectra, together with excited state lifetime measurements, to characterize their spectroscopic behavior, concentrating on the 400- to 500-nm wavelength ranges that are important in biological applications. Our data show an anomalous dependence of emission spectrum, lifetimes, and quantum yield (QY) on excitation wavelength that is particularly pronounced in the QD525. For QD525, QY values ranged from 0.2 at 480 nm excitation up to 0.4 at 450 nm and down again to 0.15 at 350 nm. For QD585, QY values were constant at 0.2 between 500 and 400 nm, but they dropped to 0.1 at 350 nm. We attribute the wavelength dependencies to heterogeneity in size and surface defects in the QD525, consistent with characteristics described previously in the chemistry literature. The results are discussed in the context of bridging the gap between what is currently known in the physical chemistry literature of quantum dots and the quantitative needs of assay development in biological applications.     

Spectroscopic characterization of a newly isolated cytochrome P450 from Rhodococcus rhodochrous.

Cytochrome P450 (P450) from Rhodococcus rhodochrous have been characterized through circular dichroism and nuclear magnetic resonance (NMR) spectroscopy, both in the substrate-free and substrate-bound forms. The data are compared with those of P450cam and indicate a close similarity of the structure of the active site in the two proteins. The substrate-free species contains low-spin iron(III), while the 2-ethoxyphenol bound species contains high-spin iron(III). The substrate is in slow exchange on the NMR time scale. The binding of CN- has been investigated and the final adduct characterized through NMR spectra. Nuclear relaxation times of the isotropically shifted signals turn out to be shorter than in other heme proteins, both in the high- and in the low-spin species. This is the result of longer electron relaxation times in P450s than in peroxidases and metmyoglobin. This property, as well as the electron paramagnetic resonance (EPR) spectrum of the substrate-free form, are discussed in terms of the presence of the cysteine as the fifth ligand of the iron ion instead of a histidine as it occurs in peroxidases and myoglobin.     

Spectroscopic and functional characterization of Cu-containing nitrite reductase from Hyphomicrobium denitrificans A3151

The Cu-containing nitrite reductase from Hyphomicrobium denitrificans (HydNIR) has been spectroscopically and functionally characterized. The visible absorption spectrum implies that the enzyme has two type 1 Cu ions in one subunit (ca. 50 kDa). The electron paramagnetic resonance (EPR) spectrum of HydNIR is simulated assuming the sum of three distinct S = 1/2 systems: two type 1 Cu signals (axial and rhombic symmetries) and one type 2 Cu signal. The intramolecular electron transfer reaction from the type 1 Cu to the type 2 Cu at pH 6.0 does not occur in the absence of nitrite, but a very slow electron transfer reaction is observed in the presence of nitrite. The apparent first-order rate constants for the intramolecular electron transfer reactions (k(ET(intra))) in the presence of nitrite and also the apparent catalytic rate constants (k(cat)) of HydNIR decrease gradually with increasing pH in the range of pH 4.5-7.5. These pH profiles are substantially similar to each other, suggesting that the intramolecular electron transfer process is linked to the subsequent nitrite reduction process.