Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.     

The Norwegian Institute of Public Health Twin Panel: a description of the sample and program of research.

The Norwegian Institute of Public Health in Oslo has an ongoing program of twin research using population-based cohorts of twins. The current database includes information on twins identified through the Medical Birth Registry of Norway and born from 1967-1979, altogether 15,370 twins. This is a longitudinal study with a cohort sequential design whereby new cohorts are recruited into the study at 5-6 year intervals. Sub-samples of these twins have participated in questionnaire studies and clinical assessment sub-projects. These projects include national and international collaborations. Our primary areas of interest include mental health and psychological well-being, obesity, asthma and allergies, health behaviors and health perceptions, comorbidity, and perinatal influences on health outcomes. This paper provides a brief overview of the data, sample, and the various research projects associated with this twin program of research.     

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.     

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein‐like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat‐inducible and appears to play a major role in Lpl1 interaction. Pre‐incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1‐Hsp90 interaction induces F‐actin formation, thus, triggering an endocytosis‐like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl‐Hsp90 interaction.     

Differences in the apical and basolateral pathways for glycosaminoglycan biosynthesis in Madin-Darby canine kidney cells

Serglycin with a green fluorescent protein tag (SG-GFP) expressed in epithelial Madin-Darby canine kidney cells is secreted mainly (85%) into the apical medium, but the glycosaminoglycan (GAG) chains on the SG-GFP protein core secreted basolaterally (15%) carry most of the sulfate added during biosynthesis (Tveit et al. (2005) J. Biol. Chem., 280, 29596-29603). Here we report further differences in apical and basolateral GAG synthesis. The less intensely sulfated chondroitin sulfate (CS) chains on apically secreted SG-GFP are longer than CS chains attached to basolateral SG-GFP, whereas the heparan sulfate (HS) chains are of similar lengths. When the supply of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is limited by chlorate treatment, the synthesis machinery maintains sulfation of HS chains on basolateral SG-GFP until it is inhibited at 50 mM chlorate, whereas basolateral CS chains lose sulfate already at 12.5 mM chlorate and become longer. Apically, incorporation of 35S-sulfate into CS is reduced to a lesser extent at higher chlorate concentrations than basolateral CS, although apical CS is less intensely sulfated than basolateral CS in control cells. Similar to what was found for basolateral HS, sulfation of apical HS was not reduced at chlorate concentrations below 50 mM. Also, protein-free, xyloside-based GAG chains secreted basolaterally are more intensely sulfated than their apical counterpart, supporting the view that separate apical and basolateral pathways exist for GAG synthesis and sulfation. Introduction of benzyl beta-d-xyloside (BX) to the GAG synthesis machinery reduces the apical secretion of SG-GFP dramatically and also the modification of SG-GFP by HS.     

The fractional contributions of elementary modes to the metabolism of Escherichia coli and their estimation from reaction entropies

The metabolism of a cell can be viewed as a weighted sum of elementary modes. Due to the multiplicity of modes the identification of the individual weights represents a non-trivial problem. To enable the determination of weighting factors we have identified and implemented two gene deletions in combination with defined growth conditions that limit the metabolism from 4374 original elementary modes to 24 elementary modes for a non-PHB synthesizing control and 40 modes for a PHB synthesizing strain. These remaining modes can be further grouped into five families that have the same overall stoichiometry. Thus, the complexity of the problem is significantly reduced, and weighting factors for each family of modes could be determined from the measurement of accumulation rates of metabolites. Moreover, it is shown that individual weights are inversely correlated with the entropy generated by the operation of the used pathways defined in elementary modes. This suggests that evolution developed cellular regulatory patterns that permit diversity of pathways while favoring efficient pathways with low entropy generation. Furthermore, such correlation provides a rational way of estimating metabolic fluxes based on the thermodynamic properties of elementary modes. This is demonstrated with an example in which experimentally determined, intracellular fluxes are shown to be highly correlated with fluxes computed based on elementary modes and reaction entropies. The analysis suggests that the set of elementary modes can be interpreted analogous to a metabolic ensemble of quantum states of a macroscopic system.     

Haemophilus influenzae surface fibrils contribute to serum resistance by interacting with vitronectin

Vitronectin inhibits the membrane attack complex of the complement system and is found both in plasma and the extracellular matrix. In this study, we have identified the outer membrane protein Haemophilus surface fibrils (Hsf) as the major vitronectin-binding protein in encapsulated H. influenzae type b. A H. influenzae mutant devoid of Hsf showed a significantly decreased binding to both soluble and immobilized vitronectin as compared with the wild-type counterpart. Moreover, Escherichia coli-expressing Hsf at the surface strongly adhered to immobilized vitronectin. Importantly, the H. influenzae Hsf mutant had a markedly reduced survival as compared with the wild-type bacterium when incubated with normal human serum. A series of truncated Hsf fragments were recombinantly manufactured in E. coli. The vitronectin binding regions were located within two separate binding domains. In conclusion, Hsf interacts with vitronectin and thereby inhibits the complement-mediated bactericidal activity, and thus is a major H. influenzae virulence factor.     

Gender comparisons between unilateral and bilateral landings

The increased number of women participating in sports has led to a higher knee injury rate in women compared with men. Among these injuries, those occurring to the ACL are commonly observed during landing maneuvers. The purpose of this study was to determine gender differences in landing strategies during unilateral and bilateral landings. Sixteen male and 17 female recreational athletes were recruited to perform unilateral and bilateral landings from a raised platform, scaled to match their individual jumping abilities. Three-dimensional kinematics and kinetics of the dominant leg were calculated during the landing phase and reported as initial ground contact angle, ranges of motion (ROM) and peak moments. Lower extremity energy absorption was also calculated for the duration of the landing phase. Results showed that gender differences were only observed in sagittal plane hip and knee ROM, potentially due to the use of a relative drop height versus the commonly used absolute drop height. Unilateral landings were characterized by significant differences in hip and knee kinematics that have been linked to increased injury risk and would best be classified as "stiff" landings. The ankle musculature was used more for impact absorption during unilateral landing, which required increased joint extension at touchdown and may increase injury risk during an unbalanced landing. In addition, there was only an 11% increase in total energy absorption during unilateral landings, suggesting that there was a substantial amount of passive energy transfer during unilateral landings.     

Deposition and canopy exchange processes in central-German beech forests differing in tree species diversity

Atmospheric deposition is an important nutrient input to forests. The chemical composition of the rainfall is altered by the forest canopy due to interception and canopy exchange. Bulk deposition and stand deposition (throughfall plus stemflow) of Na⁺, Cl^?, K⁺, Ca²⁺, Mg²⁺, PO ₄ ^3? , SO ₄ ^2? , H⁺, Mn²⁺, Al³⁺, Fe²⁺, NH ₄ ⁺ , NO ₃ ^? and N_org were measured in nine deciduous forest plots with different tree species diversity in central Germany. Interception deposition and canopy exchange rates were calculated with a canopy budget model. The investigated forest plots were pure beech (Fagus sylvatica L.) plots, three-species plots (Fagus sylvatica, Tilia cordata Mill. or T. platyphyllos Scop. and Fraxinus excelsior L.) and five-species plots (Fagus sylvatica, T. cordata or T. platyphyllos, Fraxinus excelsior, Acer platanoides L., A. pseudoplatanus L. or A. campestre L. and Carpinus betulus L.). The interception deposition of all ions was highest in pure beech plots and was negatively related to the Shannon index. The stand deposition of K⁺, Ca²⁺, Mg²⁺ and PO ₄ ^3? was higher in mixed species plots than in pure beech plots due to higher canopy leaching rates in the mixed species plots. The acid input to the canopy and to the soil was higher in pure beech plots than in mixed species plots. The high canopy leaching rates of Mn²⁺ in pure beech plots indicated differences in soil properties between the plot types. Indeed, pH, effective cation exchange capacity and base saturation were lower in pure beech plots. This may have contributed to the lower leaching rates of K⁺, Ca²⁺ and Mg²⁺ compared to the mixed species plots. However, foliar analyses indicated differences in the ion status among the tree species, which may additionally have influenced canopy exchange. In conclusion, the nutrient input to the soil resulting from deposition and canopy leaching was higher in mixed species plots than in pure beech plots, whereas the acid input was highest in pure beech plots.