Low Light Adaptation: Energy Transfer Processes in Different Types of Light Harvesting Complexes from Rhodopseudomonas palustris

Energy transfer processes in photosynthetic light harvesting 2 (LH2) complexes isolated from purple bacterium Rhodopseudomonas palustris grown at different light intensities were studied by ground state and transient absorption spectroscopy. The decomposition of ground state absorption spectra shows contributions from B800 and B850 bacteriochlorophyll (BChl) a rings, the latter component splitting into a low energy and a high energy band in samples grown under low light (LL) conditions. A spectral analysis reveals strong inhomogeneity of the B850 excitons in the LL samples that is well reproduced by an exponential-type distribution. Transient spectra show a bleach of both the low energy and high energy bands, together with the respective blue-shifted exciton-to-biexciton transitions. The different spectral evolutions were analyzed by a global fitting procedure. Energy transfer from B800 to B850 occurs in a mono-exponential process and the rate of this process is only slightly reduced in LL compared to high light samples. In LL samples, spectral relaxation of the B850 exciton follows strongly nonexponential kinetics that can be described by a reduction of the bleach of the high energy excitonic component and a red-shift of the low energetic one. We explain these spectral changes by picosecond exciton relaxation caused by a small coupling parameter of the excitonic splitting of the BChl a molecules to the surrounding bath. The splitting of exciton energy into two excitonic bands in LL complex is most probably caused by heterogenous composition of LH2 apoproteins that gives some of the BChls in the B850 ring B820-like site energies, and causes a disorder in LH2 structure.     

Single-Molecule Spectroscopy Unmasks the Lowest Exciton State of the B850 Assembly in LH2 from Rps. acidophila

We have recorded fluorescence-excitation and emission spectra from single LH2 complexes from Rhodopseudomonas (Rps.) acidophila. Both types of spectra show strong temporal spectral fluctuations that can be visualized as spectral diffusion plots. Comparison of the excitation and emission spectra reveals that for most of the complexes the lowest exciton transition is not observable in the excitation spectra due to the cutoff of the detection filter characteristics. However, from the spectral diffusion plots we have the full spectral and temporal information at hand and can select those complexes for which the excitation spectra are complete. Correlating the red most spectral feature of the excitation spectrum with the blue most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Hence, application of fluorescence-excitation and emission spectroscopy on the same individual LH2 complex allows us to decipher spectral subtleties that are usually hidden in traditional ensemble spectroscopy.     

Single-Molecule Spectroscopy Unmasks the Lowest Exciton State of the B850 Assembly in LH2 from Rps. acidophila

We have recorded fluorescence-excitation and emission spectra from single LH2 complexes from Rhodopseudomonas (Rps.) acidophila. Both types of spectra show strong temporal spectral fluctuations that can be visualized as spectral diffusion plots. Comparison of the excitation and emission spectra reveals that for most of the complexes the lowest exciton transition is not observable in the excitation spectra due to the cutoff of the detection filter characteristics. However, from the spectral diffusion plots we have the full spectral and temporal information at hand and can select those complexes for which the excitation spectra are complete. Correlating the red most spectral feature of the excitation spectrum with the blue most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Hence, application of fluorescence-excitation and emission spectroscopy on the same individual LH2 complex allows us to decipher spectral subtleties that are usually hidden in traditional ensemble spectroscopy.     

Membrane proteins of Rhodopseudomonas spheroides V. Additional chemical characterization of a pigment-lipid-associated protein isolated from chromatophores

The major protein component, Band 15, of the chromatophores of Rhodopseudomonas spheroides is associated with most of the pigments and phospholipids. The primary structure of Band 15 has been further characterized. Cyanogen bromide cleavage produced 3 oligopeptides which were present in equimolar amounts. The sum of the molecular weights of the oligopeptides derived from cyanogen bromide cleavage of Band 15 was 8600. This value compares favorably with the value of 11000 calculated from the methionine content of the protein. A C-terminal sequence, NH₂…Tyr-Ser-Glu-Glu-(Leu,Ala,Ala,Val,Val,Ala,Ala)-GlyCOOH, is proposed. A tryptic map of the protein has been obtained and the amino acid composition of each tryptic peptide determined.     

Spectral Diffusion and Electron-Phonon Coupling of the B800 BChl a Molecules in LH2 Complexes from Three Different Species of Purple Bacteria

We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the αβ-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.     

Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K_i of < 5 nM, C4A/C41A-T1 has a weaker K_i of ∼ 350 nM, and the potency of the C8A/C37A variant is further decreased to a K_i of ∼ 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.     

Identification of a novel compound that inhibits iNOS and COX-2 expression in LPS-stimulated macrophages from Schisandra chinensis

A novel α-iso-cubebenol, which has anti-inflammatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, was isolated from the fruits of Schisandra chinensis. α-iso-cubebenol inhibited LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production. Consistent with these findings, α-iso-cubebenol also reduced the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 at the protein and mRNA levels in a concentration-dependent manner. α-iso-cubebenol also inhibited LPS-induced nuclear translocation of the NF-κB p65 subunit. Furthermore, α-iso-cubebenol suppressed the phosphorylation of ERK, JNK, and p38 kinase induced by LPS. Since the novel α-iso-cubebenol blocked the production of several pro-inflammatory mediators induced by LPS in macrophages, the molecule can be useful material for the development of anti-inflammatory agents against bacterial infections or endotoxin.     

Establishment of a neonate cell line from Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) that supports replication of E. postvittana nucleopolyhedrovirus

The lightbrown apple moth (Epiphyas postvittana) is a leafroller pest that damages horticultural crops in New Zealand. This paper documents the establishment of a primary cell line from neonate E. postvittana larvae to facilitate the development of E. postvittana nucleopolyhedrovirus (EppoNPV) for control of this pest. The cell line was cultured for 36 passages and a clonal derivative designated EpN1.10 was generated that had a doubling time of 36 h at 21 °C. The EpN1.10 cell line allowed for recovery of EppoNPV from transfected genomic DNA and virus passage, as determined by occlusion body production and restriction endonuclease analysis.     

Further evidence that BALB/c and C57BL/6 gamma 2a genes originate from two distinct isotypes.

Gene conversion by the corresponding gamma 2b gene has been proposed to explain the multiple differences between the nucleic acid sequences of BALB/c (Igh-1a) and C57BL/6 (Igh-1b) gamma 2a immunoglobulin allelic genes. However, genetic analysis indicates that duplicated forms of gamma 2a genes are not only present in Eastern Asia, but also in European wild mouse populations which suggests a widespread phenomenon. In order to verify whether the gamma 2a-related isotypic genes, namely gamma 2c and gamma 2a, could correspond to those present as alleles in domestic mice (Igh-1b and Igh-1a), a genomic library from Mus m.musculus strain (MAI) was constructed. Extensive mapping of the recombinant phages and Southern blot analysis with several restriction enzymes gave the complete organization of these loci: gamma 2b (18 kb) gamma 2c (17 kb) gamma 2a (14 kb) epsilon. The homology in flanking, coding and intervening region sequences indicates that MAI gamma 2c and gamma 2a related genes correspond to C57BL/6 and BALB/c Igh-1 alleles respectively. Also, Southern blot analysis using several probes derived from exonic and intronic regions between gamma 2b and gamma 2a genes shows a 2.0- to 3.0-kb difference in the distance between gamma 2b and gamma 2a genes of BALB/c strain as compared to C57BL/6. Taken together, these results indicate that BALB/c and C57BL/6 gamma 2a genes could originate from different isotypes.     

Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.     

Purification and characterization of two extracellular peroxidases from Streptomyces sp. strain AM2, a decolorizing actinomycetes responsible for the biodegradation of natural humic acids

Two extracellular humic acids peroxidases called HaP1 and HaP2 were isolated from the Streptomyces sp. strain AM2 and, based on MALDI-TOF MS analysis. The purified enzymes were determined as monomers with molecular masses of 40,351.11 and 25,175.19 Da, respectively. The N-terminal amino acid sequences of HaP1 and HaP2 were identified, and their optimum pH values were determined as 6 and 7.5, respectively. Standard 2,4-dichlorophenol (2,4-DCP) assays showed that both enzymes had maximal activity at 55 °C. HaP2 was stable at 55 °C for more than 24 h and had a half-life of 90 min at 65 °C. Although the catalytic properties of HaP1 and HaP2 were nearly identical, their stabilities and Reinheitzahl (RZ) values were substantially different. Both peroxidases were found to be heme proteins that catalyzed the oxidation of a wide range of substrates in the presence of hydrogen peroxide (H₂O₂), with HaP2 exhibiting a broader range of substrate specificity. The characterization of peroxidase activity revealed activity against humic acids, guiacol, 2,4-DCP, l-3,4-dihydroxyphenylalanine, and 2,4,5-trichlorophenol as well as other chlorophenols in the presence of H₂O₂. However, the inhibition of peroxidase activity by the addition of potassium cyanide and sodium azide also indicated the presence of heme components in the tertiary structure of these enzymes.