Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca2+ than of Mg2+, we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca2+ induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg2+, ADP/Mg2+ or ADP/Ca2+ does not. A high strand exchange activity is observed for the filament formed with ATP/Ca2+, whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca2+ stabilizes the loop conformation and thereby the protein–DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange

High-density lipoprotein proteome dynamics in human endotoxemia

BACKGROUND: A large variety of proteins involved in inflammation, coagulation, lipid-oxidation and lipid metabolism have been associated with high-density lipoprotein (HDL) and it is anticipated that changes in the HDL proteome have implications for the multiple functions of HDL. Here, SELDI-TOF mass spectrometry (MS) was used to study the dynamic changes of HDL protein composition in a human experimental low-dose endotoxemia model. Ten healthy men with low HDL cholesterol (0.7+/-0.1 mmol/L) and 10 men with high HDL cholesterol levels (1.9+/-0.4 mmol/L) were challenged with endotoxin (LPS) intravenously (1 ng/kg bodyweight). We previously showed that subjects with low HDL cholesterol are more susceptible to an inflammatory challenge. The current study tested the hypothesis that this discrepancy may be related to differences in the HDL proteome. RESULTS: Plasma drawn at 7 time-points over a 24 hour time period after LPS challenge was used for direct capture of HDL using antibodies against apolipoprotein A-I followed by subsequent SELDI-TOF MS profiling. Upon LPS administration, profound changes in 21 markers (adjusted p-value < 0.05) were observed in the proteome in both study groups. These changes were observed 1 hour after LPS infusion and sustained up to 24 hours, but unexpectedly were not different between the 2 study groups. Hierarchical clustering of the protein spectra at all time points of all individuals revealed 3 distinct clusters, which were largely independent of baseline HDL cholesterol levels but correlated with paraoxonase 1 activity. The acute phase protein serum amyloid A-1/2 (SAA-1/2) was clearly upregulated after LPS infusion in both groups and comprised both native and N-terminal truncated variants that were identified by two-dimensional gel electrophoresis and mass spectrometry. Individuals of one of the clusters were distinguished by a lower SAA-1/2 response after LPS challenge and a delayed time-response of the truncated variants. CONCLUSIONS: This study shows that the semi-quantitative differences in the HDL proteome as assessed by SELDI-TOF MS cannot explain why subjects with low HDL cholesterol are more susceptible to a challenge with LPS than those with high HDL cholesterol. Instead the results indicate that hierarchical clustering could be useful to predict HDL functionality in acute phase responses towards LPS

Biophysical studies of DNA binding – by the large filament-forming protein Rad51 and the small minor-groove binder Hoechst\ua033258

Mechanistic insight into the nature of DNA-binding ligands is crucial for both drug development as well as understanding more complex biological reactions that take place in the cell. In this Thesis, two rather different DNA-binding molecules are considered: 1. the large, filament-forming eukaryotic recombination protein Rad51, which is essential in the strand exchange reaction during homologous recombination, the most accurate repair system of DNA double‑strand breaks, and 2. the small, synthetic DNA ligand Hoechst\ua033258, which is a model drug for DNA minor groove interactions. The Rad51 filament formation, reflected in the length of short individual Rad51 filament patches on long DNA strands, has been examined by nanofluidics in combination with fluorescence microscopy. Analyses of the dynamics of the Rad51-DNA complex in the nanochannel reveal structural variations that depend on the filament formation conditions; the choice of divalent cations (Mg2+ or Ca2+), the DNA substrate (single-\ua0or\ua0double‑stranded), and the Rad51 nucleation concentration affected the macroscopic structure of the filament. The structural effects that the divalent cations Mg2+ and Ca2+, and the accessory protein Swi5-Sfr1 exert on the Rad51-single-stranded (ss) DNA filament at a microscopic level have also been examined by linear dichroism (LD). The naturally unordered bases in ssDNA become preferentially perpendicularly oriented relative to the DNA backbone in presence of Rad51 with Ca2+ alone, or Mg2+ in combination with the accessory protein Swi5-Sfr1. A preferentially perpendicular base organization is proposed to mechanistically relate to an efficient strand exchange reaction, supposedly due to more critical base matching with the invading double‑stranded DNA. To aid future spectroscopic structural analyses of proteins that contain tyrosine residues, such as Rad51, a combined spectroscopic and in silico study of the chromophore in tyrosine has been conducted. It is demonstrated how the spectroscopic properties of tyrosine are sensitively dependent on the polarity of the environment, mainly through the ability to form hydrogen bonds and the rotation of the hydroxyl group. The last part of the Thesis deals with spectroscopic and thermodynamic studies of the binding of Hoechst\ua033258 to three different DNA oligonucleotides with AT‑tracts of various lengths. The binding at high drug‑to‑DNA ratio is especially considered, and an important conclusion is that two Hoechst\ua033258 molecules bind in parallel, a slight distance apart, in the minor groove of an oligonucleotide with 8 consecutive adenines/thymines

DNA-Complexes with Drugs and Proteins

DNA is fundamental for all living cells; the DNA holds the genetic code, which is more or less the instruction book for how all cells are built and function. Several diseases are also linked to DNA, emerging either from a mutation in our genome, which could result in a malfunctioning protein, or that the transcription of genes is somehow affected by structural changes in the DNA, induced by mutations or DNA binding molecules. Research concerning how both small molecules and larger protein assemblies bind to the DNA are therefore of great interest since these could be used as future drugs in for example gene therapy.In the first part of this Thesis the non-covalent binding to DNA of a small minor groove binder, Hoechst 33258, is examined. The molecule is rather well-studied, but there are still questions concerning its multiple binding modes to DNA sequences rich in adenines (A) and thymines (T) that remain unanswered. An increased understanding of the nature of the multiple binding modes could benefit the future design and development of sequence specific drugs. Using the thermodynamic characterization of the binding through Isothermal Titration Calorimetry (ITC) in combination with the spectroscopic properties of the formed complexes through Circular Dichroism (CD) we have analyzed the experimental results in a global dataset. We conclude that two molecules of Hoechst 33258 can bind next to each other in AT-rich sequences that consist of eight AT base pairs, but not in sequences consisting of six or less AT base pairs. They do not bind on top of one another, in the form of a sandwich, as previously proposed, nor contiguously, but with distinct separation between monomeric units.The second part of this Thesis reports how the structure and activity of the human recombination protein RAD51 (HsRad51) depends on presence of cofactors: ATP and divalent cations. The eukaryotic HsRad51 is one of the evolutionarily best-conserved proteins and homologues to it can be found in both Bacteria and Archaea. HsRad51 is involved in the strand exchange reaction of homologous recombination, which takes place during meiosis and repair of double-strand breaks in eukaryotes. With further understanding of the strand exchange reaction we might find ways to utilize it in the medicinal field, such as for correction and repair of defective genes in gene therapy, or as a potential target in cancer treatment. We confirm that the first intermediate of this reaction, in which HsRad51 forms a helical filament around a single strand of DNA, demonstrates a perpendicular organization of the DNA bases relative the filament axis when ATP and Ca2+ are present. This organization is most probably related to the observed high strand exchange activity of the HsRad51/ssDNA complex in with ATP and Ca2+. By contrast, in presence of Mg2+ we observe both poor base organization and strand exchange activity

Объединённый конгресс Европейского панкреатологического клуба и международной ассоциации панкреатологов (г. Лодзь, Республика Польша, 25-28 июня 2008 г.)

Purpose Occurrence of airway irritation among indoor swimming pool personnel was investigated. The aims of this study were to assess trichloramine exposure levels and exhaled nitric oxide in relation to the prevalence of airway symptoms in swimming pool facilities and to determine protein effects in the upper respiratory tract. Methods The presence of airway symptoms related to work was examined in 146 individuals working at 46 indoor swimming pool facilities. Levels of trichloramine, as well as exhaled nitric oxide, were measured in five facilities with high prevalence of airway irritation and four facilities with no airway irritation among the personnel. Nasal lavage fluid was collected, and protein profiles were determined by a proteomic approach. Results 17 % of the swimming pool personnel reported airway symptoms related to work. The levels of trichloramine in the swimming pool facilities ranged from 0.04 to 0.36 mg/m3. There was no covariance between trichloramine levels, exhaled nitric oxide and prevalence of airway symptoms. Protein profiling of the nasal lavage fluid showed that the levels alpha-1-antitrypsin and lactoferrin were significantly higher, and S100-A8 was significantly lower in swimming pool personnel. Conclusions This study confirms the occurrence of airway irritation among indoor swimming pool personnel. Our results indicate altered levels of innate immunity proteins in the upper airways that may pose as potential biomarkers. However, swimming pool facilities with high prevalence of airway irritation could not be explained by higher trichloramine exposure levels. Further studies are needed to clarify the environmental factors in indoor swimming pools that cause airway problems and affect the immune system

Airway irritation among indoor swimming pool personnel : trichloramine exposure, exhaled NO and protein profiling of nasal lavage fluids

Purpose Occurrence of airway irritation among indoor swimming pool personnel was investigated. The aims of this study were to assess trichloramine exposure levels and exhaled nitric oxide in relation to the prevalence of airway symptoms in swimming pool facilities and to determine protein effects in the upper respiratory tract. Methods The presence of airway symptoms related to work was examined in 146 individuals working at 46 indoor swimming pool facilities. Levels of trichloramine, as well as exhaled nitric oxide, were measured in five facilities with high prevalence of airway irritation and four facilities with no airway irritation among the personnel. Nasal lavage fluid was collected, and protein profiles were determined by a proteomic approach. Results 17 % of the swimming pool personnel reported airway symptoms related to work. The levels of trichloramine in the swimming pool facilities ranged from 0.04 to 0.36 mg/m3. There was no covariance between trichloramine levels, exhaled nitric oxide and prevalence of airway symptoms. Protein profiling of the nasal lavage fluid showed that the levels alpha-1-antitrypsin and lactoferrin were significantly higher, and S100-A8 was significantly lower in swimming pool personnel. Conclusions This study confirms the occurrence of airway irritation among indoor swimming pool personnel. Our results indicate altered levels of innate immunity proteins in the upper airways that may pose as potential biomarkers. However, swimming pool facilities with high prevalence of airway irritation could not be explained by higher trichloramine exposure levels. Further studies are needed to clarify the environmental factors in indoor swimming pools that cause airway problems and affect the immune system

UV Transition Moments of Tyrosine

To assist polarized-light spectroscopy for protein-structure analysis, the UV spectrum of p-cresol, the chromophore of tyrosine, was studied with respect to transition moment directions and perturbation by solvent environment. From linear dichroism (LD) spectra of p-cresol aligned in stretched matrices of poly(vinyl alcohol) and polyethylene, the lowest pi-pi* transition (L-b) is found to have pure polarization over its entire absorption (250-300 nm) with a transition moment perpendicular to the symmetry axis (C-1-C-4), both in polar and nonpolar environments. For the second transition (L-a), polarized parallel with the symmetry axis, a certain admixture of intensity with orthogonal polarization is noticed, depending on the environment. While the L-b spectrum in cydohexane shows a pronounced vibrational structure, it is blurred in methanol, which can be modeled as due to many microscopic polar environments. With the use of quantum mechanical (QM) calculations, the transition moments and solvent effects were analyzed with the B3LYP and omega B97X-D functionals in cyclohexane, water, and methanol using a combination of implicit and explicit solvent models. The blurred L-b band is explained by solvent hydrogen bonds, where both accepting and donating a hydrogen causes energy shifts. The inhomogeneous solvent-shift sensitivity in combination with robust polarization can be exploited for analyzing tyrosine orientation distributions in protein complexes using LD spectroscopy

Minor-Groove Binding Drugs: Where Is the Second Hoechst 33258 Molecule?

Hoechst 33258 binds with high affinity into the minor groove of AT-rich sequences of double-helical DNA. Despite extensive studies of this and analogous DNA binding molecules, there still remains uncertainty concerning the interactions when multiple ligand molecules are accommodated within close distance. Albeit not of direct concern for most biomedical applications, which are at low drug concentrations, interaction studies for higher drug binding are important as they can give fundamental insight into binding mechanisms and specificity, including drug self-stacking interactions that can provide base-sequence specificity. Using circular dichroism (CD), isothermal titration calorimetry (ITC), and proton nuclear magnetic resonance (1H NMR), we examine the binding of Hoechst 33258 to three oligonucleotide duplexes containing AT regions of different lengths: [d(CGCGAATTCGCG)]2 (A2T2), [d(CGCAAATTTGCG)]2 (A3T3), and [d(CGAAAATTTTCG)]2 (A4T4). We find similar binding geometries in the minor groove for all oligonucleotides when the ligand-to-duplex ratio is less than 1:1. At higher ratios, a second ligand can be accommodated in the minor groove of A4T4 but not A2T2 or A3T3. We conclude that the binding of the second Hoechst to A4T4 is not cooperative and that the molecules are sitting with a small separation apart, one after the other, and not in a sandwich structure as previously proposed

Lifetime Heterogeneity of DNA-Bound dppz Complexes Originates from Distinct Intercalation Geometries Determined by Complex-Complex Interactions

Despite the extensive interest in structurally explaining the photophysics of DNA-bound [Ru(phen)(2)dppz](2+) and [Ru(bpy)(2)dppz](2+), the origin of the two distinct emission lifetimes of the pure enantiomers when intercalated into DNA has remained elusive. In this report, we have combined a photophysical characterization with a detailed isothermal titration calorimetry study to investigate the binding of the pure Delta and Lambda enantiomers of both complexes with [poly(dAdT)](2). We find that a binding model with two different binding geometries, proposed to be symmetric and canted intercalation from the minor groove, as recently reported in high-resolution X-ray structures, is required to appropriately explain the data. By assigning the long emission lifetime to the canted binding geometry, we can simultaneously fit both calorimetric data and the binding-density-dependent changes in the relative abundance of the two emission lifetimes using the same binding model. We find that all complex complex interactions are slightly unfavorable for Delta-[Ru(bpy)(2)dppz](2+), whereas interactions involving a complex canted away from a neighbor are favorable for the other three complexes. We also conclude that Delta-[Ru(bpy)(2)dppz](2+) preferably binds isolated, Delta-[Ru(phen)(2)dppz](2+) preferably binds as duplets of canted complexes, and that all complexes are reluctant to form longer consecutive sequences than triplets. We propose that this is due to an interplay of repulsive complex complex and attractive complex-DNA interactions modulated by allosteric DNA conformation changes that are largely affected by the nature of the ancillary ligands