A method has been developed that combines electrospray ionization mass spectrometry with pH control to provide analysis of metals in native or reconstituted metallothioneins. These metalloproteins cooperatively bind seven divalent metal ions, most commonly Zn2+ and Cd2+. Since the protein is denatured and metal ions are lost below pH3, the pH of the electrospray solution is critical to successful results. The metal-free apoprotein was detected with its most abundant ions in a charge state of 6+, while the folded metallothionein-metal complexes were observed with lower charge states. The retention of seven metals in the molecular ions detected is consistent with the hypothesis that metallothionein retains its conformation in the gas phase. This mass spectrometric technique can be used to determine rapidly and accurately how many and what cations are incorporated per molecule of protein. Information about molar distributions and estimates of relative abundances of various complexes in the sample can be acquired in a single measurement.

Fimbrial adhesins of pathogenic bacteria are linear protein associates responsible for binding to the specific host cell receptors. They are assembled via the chaperone/usher pathway conserved in Gram-negative bacteria. These adhesive organelles are characterized by the high resistance to dissociation and unfolding caused by temperature or chemical denaturants. The self-complemented (SC) recombinant subunits of adhesive structures make up the minimal model used to analyze stability phenomena of these organelles. The SC subunits are both highly stabilized thermodynamically and kinetically. They are characterized by a standard free energy of unfolding of 70-80 kJ/mol and a rate constant of unfolding of 10(-17) s(-1) (half-life of unfolding of 10(8) years at 25 degrees C). The DraE subunit of Dr fimbriae is characterized by a disulfide bond that joins the beginning of the A1 strand with the end of the B strand. Such localization is unique and differentiates this protein from other proteins of the Ig-like family. Sequence analysis shows that many protein subunits of adhesive structures possess cysteines that may form a potential disulfide bond homologous to that of DraE. In this paper, we investigate the influence of this noncanonical disulfide bond on the stability of DraE-sc by constructing a DraE-sc-DeltaSS mutant protein (Cys/Ala mutant). This construct unfolds thermally at a T(m) of 65.4 degrees C, more than 20 degrees C lower than that of the native DraE-sc protein, and possesses a different unfolding mechanism. The calculated standard free energy of unfolding of DraE-sc-DeltaSS is equal to 30 +/- 5 kJ/mol. This allows us to suggest that the disulfide bond is an important stabilizing feature of many fimbrial subunits.

Abstract:We have developed a simple docking procedure that is able to utilize low-resolution models of proteins created by structure prediction algorithms such as threading or ab initio folding to predict the conformation of receptor-small ligand complexes. In our approach, using only approximate, discretized models of both molecules, we search for the steric and quasi-chemical complementarity between a ligand and the receptor molecules. This averaging procedure allows for the compensation of numerous structural inaccuracies resulting from the theoretical predictions of the receptor structure. The best relative orientation of these two models is obtained by an exhaustive scan over the rigid body's six-dimensional translational and rotational degrees of freedom. The search method is based on a real space grid-searching algorithm, unlike docking methods based on the fast Fourier Transform algorithm. We have applied this algorithm to rebuild structures of several complexes available in the Protein Data Bank. The structures of the receptors are produced by means of our threading algorithm PROSPECTOR, subsequently refined, and then utilized in the docking experiment. In many cases, not only is the localization of the binding site on the receptor surface correctly identified, but the proper orientation of the bounded ligand is also reasonably well reproduced within the level of accuracy of the modeled receptor itself.

In this paper, the hydration of a model protein--hen egg white lysozyme in aqueous solution has been presented. The leading method used was FTIR spectroscopy with an application of a technique of semi-heavy water (HDO) isotope dilution. Analysis of spectra of HDO isotopically diluted in water solution of lysozyme allowed us to isolate HDO spectra affected by lysozyme, and thus to characterise the energetic state of water molecules and their arrangement around protein molecules. The number of water molecules and the shape of the affected HDO spectrum were obtained using a classical and a chemometric method. This shape showed that the HDO spectrum affected by lysozyme may be presented as a superposition of two spectra corresponding to HDO affected by N-methylacetamide and the carboxylate anion (of the formic acid). Moreover, based on the difference in intermolecular distances distribution of water molecules (obtained from spectral data), we demonstrated that the lysozyme molecule causes a decrease in population of weak hydrogen bonds, and concurrently increases the probability of an occurrence of short hydrogen bonds in water affected by lysozyme. This conclusion was also confirmed by the molecular dynamics (MD) simulation.

Square-wave anodic stripping voltammetry (SWASV) of nanomolar concentration levels of lead and calcium at glassycarbon-based thin mercury film electrodes was Investigated In the presence of dissolved oxygen. Anodic stripping measurements In nondeaerated solutions were possible because of the depletion of oxygen at the electrode surface prior to the stripping step and because of the fast-scanning ability of square-wave voltammetry, which allows for the completion of the stripping step before any significant oxidation of the amalgam by diffusing oxygen can occur. Similar measurements by differential pulse anodic stripping voltammetry are not possible unless oxygen Is removed from solution. Thin mercury films showed good stability In the presence of dissolved oxygen. Linear calibration curves were obtained for lead and cadmium In acidic media (HCI and acetate buffer, pH 4.8), with no significant loss In stripping current sensitivity due to dissolved oxygen. The SWASV method was successfully used for the determination of lead In untreated, air-saturated tap water samples containing added 0.01 M HCI. The determinations by SWASV agreed very well with the results obtained by electrothermal atomic absorption spectrophotometry. With the time-consuming solution purging step absent and the very fast stripping step, the deposition step Is the only time-limiting factor of the method.

BackgroundThe global spread of bacterial resistance has given rise to a growing interest in new anti-bacterial agents with a new strategy of action. Pilicides are derivatives of ring-fused 2-pyridones which block the formation of the pili/fimbriae crucial to bacterial pathogenesis. They impair by means of a chaperone-usher pathway conserved in the Gram-negative bacteria of adhesive structures biogenesis. Pili/fimbriae of this type belong to two subfamilies, FGS and FGL, which differ in the details of their assembly mechanism. The data published to date have shown that pilicides inhibit biogenesis of type 1 and P pili of the FGS type which are encoded by uropathogenic E. coli strains.ResultsWe evaluated the anti-bacterial activity of literature pilicides as blockers of the assembly of a model example of FGL-type adhesive structures, – the Dr fimbriae encoded by a dra gene cluster of uropathogenic Escherichia coli strains. In comparison to the strain grown without pilicide, the Dr+ bacteria cultivated in the presence of the 3.5 mM concentration of pilicides resulted in a reduction of 75 to 87% in the adherence properties to CHO cells expressing Dr fimbrial DAF receptor protein. Using quantitative assays, we determined the amount of Dr fimbriae in the bacteria cultivated in the presence of 3.5 mM of pilicides to be reduced by 75 to 81%. The inhibition effect of pilicides is concentration dependent, which is a crucial property for their use as potential anti-bacterial agents. The data presented in this article indicate that pilicides in mM concentration effectively inhibit the adherence of Dr+ bacteria to the host cells, – the crucial, initial step in bacterial pathogenesis.ConclusionsStructural analysis of the DraB chaperone clearly showed it to be a model of the FGL subfamily of chaperones. This permits us to conclude that analyzed pilicides in mM concentration are effective inhibitors of the assembly of adhesins belonging to the Dr family, and more speculatively, of other FGL-type adhesive organelles. The presented data and those published so far permit to speculate that based on the conservation of chaperone-usher pathway in Gram-negative bacteria , the pilicides are potential anti-bacterial agents with activity against numerous pathogens, the virulence of which is dependent on the adhesive structures of the chaperone-usher type.

BackgroundIn recent years, there has been an increasing interest in SSBs because they find numerous applications in diverse molecular biology and analytical methods.ResultsWe report the characterization of single-stranded DNA binding proteins (SSBs) from the thermophilic bacteria Thermotoga maritima (TmaSSB) and Thermotoga neapolitana (TneSSB). They are the smallest known bacterial SSB proteins, consisting of 141 and 142 amino acid residues with a calculated molecular mass of 16.30 and 16.58 kDa, respectively. The similarity between amino acid sequences of these proteins is very high: 90% identity and 95% similarity. Surprisingly, both TmaSSB and TneSSB possess a quite low sequence similarity to Escherichia coli SSB (36 and 35% identity, 55 and 56% similarity, respectively). They are functional as homotetramers containing one single-stranded DNA binding domain (OB-fold) in each monomer. Agarose mobility assays indicated that the ssDNA-binding site for both proteins is salt independent, and fluorescence spectroscopy resulted in a size of 68 ± 2 nucleotides. The half-lives of TmaSSB and TneSSB were 10 h and 12 h at 100°C, respectively. When analysed by differential scanning microcalorimetry (DSC) the melting temperature (Tm) was 109.3°C and 112.5°C for TmaSSB and TneSSB, respectively.ConclusionThe results showed that TmaSSB and TneSSB are the most thermostable SSB proteins identified to date, offering an attractive alternative to TaqSSB and TthSSB in molecular biology applications, especially with using high temperature e. g. polymerase chain reaction (PCR).

No abstract