Fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases. Application to subtilisins.

A fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases is described. It consists of: selective labelling of the corresponding subsites with a fluorescent group by using N alpha-dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ated peptide chloromethanes containing different numbers of amino acid residues, and probing the immediate environment of the subsites by quenching experiments using ionic and neutral quenchers. Intramolecular distances between the subsites and particular chromophores can be also determined. The technique is of general applicability to all serine proteinases. The above mentioned approach was applied to two proteinases: subtilisin Novo and mesentericopeptidase. It was concluded that the substrate-binding site of mesentericopeptidase is considerably more polar than that of subtilisin Novo. Intramolecular distances between the labelled subsites and tryptophan residues in the two proteinases were determined.     

Factors affecting the dissociation and aggregation of human interferon gamma

The biologically active form of interferon γ (IFN-γ) is a dimer consisting of two identical non-covalently bound polypeptide chains. We have studied spectroscopically the dimer–monomer dissociation equilibrium of human recombinant IFN-γ and have found that the monomers possess approximately 50% lower Trp quantum yield than the dimers [Boteva et al. Biochemistry 1996;35:14825]. In the present study we characterise the conformational properties of the two states — monomeric and dimeric, and analyse the effects of the salt composition of human blood plasma, physiological cations K⁺, Na⁺, Ca²⁺ and Mg²⁺ and mechanical stress on the dimer–monomer equilibrium. A medium with electrolyte composition of human blood plasma increases both the association and dissociation rate constants without shifting significantly the dimer–monomer equilibrium. The physiological cations shift the equilibrium towards dissociation of dimers into monomers by lowering the activation energy and the free energy of the process thus decreasing the stability of IFN-γ. Mechanical stress caused by stirring of the protein solution reduces irreversibly the Trp fluorescence by 75–80% and decreases significantly the -helical content and favours the aggregation.     

Acid phosphatase distribution and localization in the fungus Humicola lutea

Acid phosphatase activities in a culture liquid and mycelial extract were studied in submerged cultures of the filamentous fungus Humicola lutea 120-5 in casein-containingmedia with and without inorganic phosphate (Pi). The Pi-repressible influence on the phosphatase formation was demonstrated. Significant changes in the distribution of acid phosphatase between the mycelial extract and culture liquid were observed at the transition of the strain from exponential to stationary phase. Some differences in the cytochemical localization of phosphatase in dependence of Pi in the media and the role of the enzyme in the release of available phosphorus from the phosphoprotein casein for fungal growth were discussed.     

Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli

Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, alpha-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids.     

PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones

The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell-cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants (K(d)), which are similar to the previously determined K(d) for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).     

Evaluation and mapping of the conservation significance of habitats using GIS: an example from Crete, Greece

To ensure the long-term future of NATURA 2000 sites across Europe, effective techniques are required for evaluating and monitoring their conservation significance. This paper describes a GIS-based method that uses multi-criteria evaluation (MCE) to determine the conservation significance of vegetation communities and habitats for a case study of a proposed NATURA 2000 site on the northwest coast of Crete, Greece. The method uses the most frequently used criteria for the selection of priority areas for nature conservation—species and habitat diversity, rarity of species and habitats, naturalness, threat of human disturbance and replaceability. For each community and corresponding habitat type, each criterion was scored according to field data and expert knowledge using a numerical scale. The final conservation score for each community was derived using MCE within a GIS and mapped. The results demonstrated that the method is an effective tool for evaluating and comparing conservation significance and could be applied to other sites across Europe and to monitor change.     

Comparison of some kinetic parameters of peroxidase and IAA oxidase in the course of growth and differentiation of plant cells

An attempt is made to characterize the functional activity of the protein moleculo possessing both peroxidase and IAA oxidase activity by comparing the kinetic parameters for the two types of enzyme activity with regard to the following substrates: H₂O₂, benzidine, guaiacol and IAA. The curves expressing the dependence of the enzyme reaction velocity on the concentration of the enzyme or the substrate are different depending on the enzyme extract origin and the type of the substrate. It is established that the K_m of peroxidase for IAA decreases while its K_m for H₂O₂ increases during cell development. Both types of enzyme activity show similar pH and temperature dependence. The presented data show that IAA oxidase activity of the peroxidase develops as extension and differentiation of the root cells proceed. This is one of the possible mechanisms through which peroxidase may participate in the regulation of growth and differentiation of the primary root cells of maize (Zea mays L.)     

Cystic fibrosis mutations and associated haplotypes in Bulgaria – a comparative population genetic study

We present data on the population genetics of cystic fibrosis (CF) in Bulgaria, obtained by comprehensive mutation analysis and the construction of intragenic microsatellite haplotypes. The sample of 262 CF alleles analysed is representative of the patients diagnosed during the period of referral and of the three main ethnic groups in the country. ΔF508 accounted for 100% of Gypsy CF alleles, which thus differed significantly from both Bulgarians and ethnic Turks. In Bulgarian and Turkish CF patients, 92% of the mutant alleles were identified, yielding a total of 25 different mutations, of which only 7 occurred at frequencies higher than 1%. The findings were compared to other European populations and to the distribution of phenylketonuria mutations. Genetic distances and population trees demonstrated that in the south-eastern tip of Europe, the overall distribution of CF mutations and polymorphic haplotypes is very close to that of Mediterranean populations, with a high frequency of N1303K and G542X, a large number of rare mutations and a prevalence of the 23 31 13 haplotype in association with ΔF508. These findings are consistent with a main role for the Neolithic expansion in the shaping of the CF mutation spectrum in Bulgaria and southern Europe. Received: 1 September 1996     

Conformational transitions of proteins engaged in DNA double-strand break repair, analysed by tryptophan fluorescence emission and FRET

We analysed protein-DNA and protein-protein interactions relevant to the repair of DNA DSBs (double-strand breaks) by NHEJ (non-homologous end-joining). Conformational transitions in mammalian DNA ligases III (LigIII) and IV (LigIV), as well as in PARP-1 [poly(ADP-ribose) polymerase-1], were analysed upon binding to double-stranded DNA by changes in tryptophan emission and FRET (F?rster resonance energy transfer) from tryptophan to DNA-conjugated Alexa Fluor? 532. For LigIII, two non-equivalent high- and low-affinity DNA-binding sites are detected interacting sequentially with DNA. PARP-1 displays a single high-affinity DNA-binding site and can displace bound DNA fragments from the low-affinity site of LigIII, consistent with its mediator role in LigIII-DNA interactions. For the LX [LigIV-XRCC4 (X-ray cross-complementation group 4)] complex, a single DNA-binding site is detected. Binding of Ku to DNA was accompanied by conformational changes in the protein and intermolecular FRET from dansyl chromophores of the labelled Ku to the Alexa Fluor? chromophores of Alexa Fluor? 532-conjugated DNA. The average distance of 5.7?nm calculated from FRET data is consistent with a location of Ku at the very end of the DNA molecule. Binding of LX to Ku-DNA complexes is associated with conformational changes in Ku, translocating the protein further towards the DNA ends. The protein-protein and protein-DNA interactions detected and analysed generate a framework for the characterization of molecular interactions fundamental to the function of NHEJ pathways in higher eukaryotes.