Biomedical subjects

Jesús M Sanz

Publications and source records attributed to Jesús M Sanz.

9 recordsLinked to original sources

PubMed2007

Extensive unfolding of the C-LytA choline-binding module by submicellar concentrations of sodium dodecyl sulphate.

We have investigated the stability of the choline-binding module C-LytA against sodium dodecyl sulphate (SDS)-induced unfolding at pH 7.0 and 20 degrees C. A major intermediate with an unfolded N-terminal region accumulates at around 0.75 mM SDS, whereas 2.0 mM SDS was sufficient for a complete unfolding. This might be the first report of a protein being extensively unfolded by submicellar concentrations of SDS, occurring through formation of detergent clusters on the protein surface. All transitions were reversible upon SDS complexation with beta-cyclodextrin, allowing the calculation of thermodynamic parameters. A model for the unfolding of C-LytA by SDS is presented and compared to a previous denaturation scheme by guanidine hydrochloride.

Bacterial Proteins↗

PubMed2007

Inhibition of pneumococcal choline-binding proteins and cell growth by esters of bicyclic amines.

Streptococcus pneumoniae is one of the major pathogens worldwide. The use of currently available antibiotics to treat pneumococcal diseases is hampered by increasing resistance levels; also, capsular polysaccharide-based vaccination is of limited efficacy. Therefore, it is desirable to find targets for the development of new antimicrobial drugs specifically designed to fight pneumococcal infections. Choline-binding proteins are a family of polypeptides, found in all S. pneumoniae strains, that take part in important physiologic processes of this bacterium. Among them are several murein hydrolases whose enzymatic activity is usually inhibited by an excess of choline. Using a simple chromatographic procedure, we have identified several choline analogs able to strongly interact with the choline-binding module (C-LytA) of the major autolysin of S. pneumoniae. Two of these compounds (atropine and ipratropium) display a higher binding affinity to C-LytA than choline, and also increase the stability of the protein. CD and fluorescence spectroscopy analyses revealed that the conformational changes of C-LytA upon binding of these alkaloids are different to those induced by choline, suggesting a different mode of binding. In vitro inhibition assays of three pneumococcal, choline-dependent cell wall lytic enzymes also demonstrated a greater inhibitory efficiency of those molecules. Moreover, atropine and ipratropium strongly inhibited in vitro pneumococcal growth, altering cell morphology and reducing cell viability, a very different response than that observed upon addition of an excess of choline. These results may open up the possibility of the development of bicyclic amines as new antimicrobials for use against pneumococcal pathologies.

Amines↗

PubMed2005

Accumulation of partly folded states in the equilibrium unfolding of the pneumococcal choline-binding module C-LytA.

Choline-binding modules are present in some virulence factors and many other proteins of Streptococcus pneumoniae (Pneumococcus). The most extensively studied choline-binding module is C-LytA, the C-terminal moiety of the pneumococcal cell-wall amidase LytA. The three-dimensional structure of C-LytA is built up from six loop-hairpin structures forming a left-handed beta-solenoid with four choline-binding sites. The affinity of C-LytA for choline and other structural analogues allows its use as an efficient fusion tag for single-step purification of hybrid proteins. In the present study, we characterize the folding and stability of C-LytA by chemical and thermal equilibrium denaturation experiments. Unfolding experiments using guanidinium chloride at pH 7.0 and 20 degrees C suggest the existence of two partly folded states (I1 and I2) in the following model: N (native)-->I1<=>I2. The N-->I1 transition is non-co-operative and irreversible, and is significant even in the absence of a denaturant. In contrast, the I1<=>I2 transition is co-operative and reversible, with an associated freeenergy change (DeltaG(0)) of 30.9+/-0.8 kJ x mol(-1). The residual structure in the I2 state is unusually stable even in 7.4 M guanidinium chloride. Binding of choline stabilizes the structure of the native state, induces its dimerization and prevents the accumulation of the I1 species ([N]2<=>[I2]2, DeltaG(0)=50.1+/-0.8 kJ x mol(-1)). Fluorescence and CD measurements, gel-filtration chromatography and limited proteolysis suggest that I1 differs from N in the local unfolding of the N-terminal beta-hairpins, and that I2 has a residual structure in the C-terminal region. Thermal denaturation of C-LytA suggests the accumulation of at least the I1 species. These results might pave the way for an effective improvement of its biotechnological applications by protein engineering.

Choline↗

PubMed2003

Molecular determinants of the hpa regulatory system of Escherichia coli: the HpaR repressor.

The HpaR-mediated regulation of the hpa-meta operon (Pg promoter) of the 4-hydroxyphenylacetic acid catabolic pathway of Escherichia coli has been studied. The HpaR regulator was purified to homogeneity showing that it is able to bind selectively to 4-hydroxyphenylacetic, 3-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids, which act as inducers of the system. The role of HpaR as a repressor and the requirement for cAMP receptor protein for maximal activity have been confirmed by in vitro transcription analyses. Two DNA operators, OPR1 and OPR2, have been identified in the intergenic region located between the hpa-meta operon and the hpaR gene. The OPR1 operator contains a perfect palindromic sequence overlapping the transcriptional +1 start site of the Pg promoter. The OPR2 operator shows a similar but imperfect palindromic sequence and is located far downstream of the +1 start site of the Pr promoter. The binding of HpaR to OPR2 displays a clear cooperativity with OPR1 binding. Based on the above observations and the results of permanganate footprinting experiments, a repression mechanism for HpaR is postulated. A 3-dimensional model of HpaR, generated by comparison with the crystal structures of the homologous regulators, MarR and MexR, suggests that HpaR is a dimer that contains a typical winged-helix DNA binding motif in each subunit.

Amino Acid Sequence↗

PubMed2003

Modulation of pPS10 host range by plasmid-encoded RepA initiator protein.

We report here the isolation and analysis of novel repA host range mutants of pPS10, a plasmid originally found in Pseudomonas savastanoi. Upon hydroxylamine treatment, five plasmid mutants were selected for their establishment in Escherichia coli at 37 degrees C, a temperature at which the wild-type form cannot be established. The mutations were located in different functional regions of the plasmid RepA initiation protein, and the mutants differ in their stable maintenance, copy number, and ability to interact with sequences of the basic replicon. Four of them have broadened their host range, and one of them, unable to replicate in Pseudomonas, has therefore changed its host range. Moreover, the mutants also have increased their replication efficiency in strains other than E. coli such as Pseudomonas putida and Alcaligenes faecalis. None of these mutations drastically changed the structure or thermal stability of the wild-type RepA protein, but in all cases an enhanced interaction with host-encoded DnaA protein was detected by gel filtration chromatography. The effects of the mutations on the functionality of RepA protein are discussed in the framework of a three-dimensional model of the protein. We propose possible explanations for the host range effect of the different repA mutants, including the enhancement of limiting interactions of RepA with specific host replication factors such as DnaA.

Bacterial Proteins↗

PubMed2002

Hints of nonhierarchical folding of acidic fibroblast growth factor.

We have analyzed by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) the helical propensity of the all-beta protein acidic fibroblast growth factor (aFGF) and two peptides corresponding to beta-strand 8 (beta8 peptide, amino acids 95-107) and the beta-strand 8/turn/beta-strand 9 hairpin (beta8/9 peptide, amino acids 95-114), which has been involved in receptor binding. A secondary structure prediction of aFGF carried out by several procedures labels the 95-104 sequence as predominantly alpha-helical. A titration of aFGF with 2,2,2-trifluoroethanol (TFE) induces a change in the far-UV CD spectrum of the protein giving rise to a prominent alpha-helical shape (22% alpha-helix). The cooperativity of the transition and the moderate TFE concentrations used (midpoint at 24%) suggest that the effect of TFE is specific. Moreover, a titration performed at pH 2 yields a higher amount of alpha-helix (55%) at a smaller TFE concentration. Synthetic peptides containing the beta8 and beta8/9 sequences display a random coil conformation at pH 7 but acquire alpha-helical structure in the presence of TFE, methanol, and SDS micelles. At pH below 3.0 a significant amount (20-30%) of alpha-helical conformation is present in both the beta8 and beta8/9 peptides even in the absence of other solvent additives. The secondary structure of the peptides was determined by proton nuclear magnetic resonance (1H NMR). These results suggest that the 95-114 sequence of aFGF has helical propensity and that the protein may fold nonhierarchically in the early steps of folding, acquiring its final beta-structure by a later interaction with the rest of the polypeptide.

Amino Acid Sequence↗

PubMed2002

Folding of dimeric methionine adenosyltransferase III: identification of two folding intermediates.

Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III.

Anilino Naphthalenesulfonates↗

PubMed2002

Modulation of pPS10 host range by DnaA.

Narrow-host-range plasmid pPS10, originally found in Pseudomonas savastanoi, is unable to replicate in other strains such as Escherichia coli. Here, we report that the establishment of pPS10 in E. coli can be achieved by a triple mutation in the dnaA gene of E. coli (dnaA403), leading to Q14amber, P297S and A412V changes in the DnaA host replication protein (DnaA403 mutant). As the E. coli strain used contained double amber suppressor mutations (supE, supF), the amber codon in dnaA403 can be translated into glutamine or tyrosine. Genetic analysis of DnaA proteins containing either the individual changes or their different combinations suggests that the P297S mutation is crucial for the establishment of the pPS10 replicon in E. coli. The data also indicate that the P297S change is toxic to the cell and that the additional mutations in DnaA403 could contribute to neutralize this toxicity. To our knowledge, this work reports the first chromosome mutant described in the literature that allows the host range broadening of a plasmid, highlights the essential role played by DnaA in the establishment of pPS10 replicon in E. coli and provides support for the hypothesis that interactions between RepA and DnaA modulate the establishment of pPS10 in that bacteria and probably in other species.

Bacterial Proteins↗

PubMedDate not supplied

Rapid urease test utility for Helicobacter pylori infection diagnosis in gastric ulcer disease.

BACKGROUND/AIMS: To establish rapid urease test utility for initial diagnosis of Helicobacter pylori infection in gastric ulcer patients and to determine the best site for sampling for gastric biopsies. METHODOLOGY: Seventy consecutive gastric ulcer patients were prospectively studied. All these patients underwent three biopsies from both antrum and body (two for hematoxylin-eosin staining and one for rapid urease test -Jatrox H. p. Test-). Likewise, IgG ELISA serology and 13C-urea breath test were carried out. Gold standard for H. pylori infection was defined as two or more tests (i.e., histology, serology, breath test) with positive results. RESULTS: Rapid urease test yielded 96.8% sensitivity (95% CI = 89-99%) and 100% (66-100%) specificity when using biopsy specimens from the body, with identical results when biopsy specimens from both antrum and body were considered together. However, when only biopsy specimens from the antrum were used, sensitivity dropped to 72.6% (60-82%) and specificity was 100% (66-100%). As far as concordance between rapid urease test and histology is concerned, we found a "proportion of positive agreement" of 0.78 for the antrum, with 0.46 kappa statistic (P < 0.0001) and 15 McNemar statistic (P < 0.0001). For the gastric body, "proportion of positive agreement" was 0.98, with 0.94 kappa statistic (P < 0.0001) and 1 McNemar statistic (P = 0.3). Larger (P < 0.01) prevalence of both glandular atrophy (17.8%, 11-28%) and intestinal metaplasia (68.5%, 57-78%) was observed in the antrum in comparison with that in the body (4.1%, 1-11%; and 16.4%, 10-26%, respectively). CONCLUSIONS: Biopsy specimens from the body should always be obtained when the rapid urease test is performed to diagnose H. pylori infection in gastric ulcer, since this procedure is less accurate when biopsy specimens from the antrum are used, probably due to larger prevalence of both glandular atrophy and intestinal metaplasia in the latter site. Likewise, it seems that rapid urease test from body biopsies is sufficient to reach a reliable infection diagnosis in gastric ulcer patients as this procedure performed with antrum biopsies fails to improve its overall results.

Aged↗