47 research outputs found
The dimeric assembly of Photobacterium leiognathi and Salmonella typhimurium SodC1 Cu,Zn superoxide dismutases is affected differently by active site demetallation and pH: An analytical ultracentrifuge study
No full textTo establish whether the species-specific variations at the subunit interface of bacterial Cu,Zn superoxide dismutases affect dimer assembly, the association state of the Photobacterium leiognathi (PISOD) and Salmonella typhimurium (StSOD) enzymes, which differ in 11 out of 19 interface residues, was investigated by analytical ultracentrifugation. The same linkage pattern correlates quaternary assembly, active site metallation, and pH in the two enzymes albeit with quantitative differences. Both holo-enzymes are stable dimers at pH 6.8 and 8.0, although their shape is altered at alkaline pH. In contrast, dimer stability is affected differently by metal removal. Thus, apo-StSOD is a stable dimer at pH 6.8 whereas apo-PISOD is in reversible monomer-dimer equilibrium. In both apoproteins a pH increase to 8.0 favors monomerization. These effects prove the existence of long-range communication between the active site and the subunit interface and provide a structural explanation for the known functional differences between the two enzymes. (c) 2007 Elsevier Inc. All rights reserved
Unusually strong H-bonding to the heme ligand and fast geminate recombination dynamics of the carbon monoxide complex of Bacillus subtilis truncated hemoglobin.
No full textThe active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been
characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO
rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance
Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at
545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were
observed in infrared absorption and resonance Raman measurements. The very low C-O stretching
frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1)
indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison
with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific
interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogenbonding
network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489
cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the
CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis.
Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong
H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization
A novel chimera: the "truncated hemoglobin-antibiotic monooxygenase" from Streptomyces avermitilis.
No full textNovel chimeric proteins made of a globin domain fused with a "cofactor free" monooxygenase domain have been identified within the Streptomyces avermitilis and Frankia sp. genomes by means of bioinformatics methods. Structure based sequence alignments show that the globin domains of both proteins can be unambiguously assigned to the truncated hemoglobin family, in view of the, striking similarity to the truncated hemoglobins from Mycobacterium tuberculosis, Thermobifida fusca and Bacillus subtilis. In turn, the non-heme domains belong to a family of small (about 100 aminoacids) homodimeric proteins annotated as antibiotic biosynthesis monooxygenases, despite the lack of a cofactor (e.g., a metal, a flavin or a heme) necessary for oxygen activation.
The chimeric protein from S. avermitilis has been cloned, expressed and characterized. The protein is a stable dimer in solution based on analytical ultracentrifugation experiments. The heme ligand binding properties with oxygen and carbonmonoxide resemble those of other Group If truncated hemoglobins. In addition, an oxygen dependent redox activity has been demonstrated towards easily oxidizable substrates such as menadiol and p-aminophenol. These findings suggest novel functional roles of truncated hemoglobins, which might represent a vast class of multipurpose oxygen activating/scavenging proteins whose catalytic action is mediated by the interaction with cofactor free monooxygenases. (c) 2007 Elsevier B.V. All rights reserved
The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase
No full textPyridoxal 5'-phosphate-dependent enzymes may be grouped into five structural superfamilies of proteins, corresponding to as many fold types. The fold type I is by far the largest and most investigated group. An important feature of this fold, which is characterized by the presence of two domains, appears to be the existence of three clusters of evolutionarily conserved hydrophobic contacts. Although two of these clusters are located in the central cores of the domains and presumably stabilize their scaffold, allowing the correct alignment of the residues involved in cofactor and substrate binding, the role of the third cluster is much less evident. A site-directed mutagenesis approach was used to carry out a model study on the importance of the third cluster in the structure of a well characterized member of the fold type I group, serine hydroxymethyltransferase from Escherichia coli. The experimental results obtained indicated that the cluster plays a crucial role in the stabilization of the quaternary, native assembly of the enzyme, although it is not located at the subunit interface. The analysis of the crystal structure of serine hydromethyltransferase suggested that this stabilizing effect may be due to the strict structural relation between the cluster and two polypeptide loops, which, in fold type I enzymes, mediate the interactions between the subunits and are involved in cofactor binding, substrate binding and catalysis
Organization and evolution of Gorilla centromeric DNA from old strategies to new approaches
No full textThe centromere/kinetochore interaction is responsible for the pairing and segregation of replicated chromosomes in eukaryotes. Centromere DNA is portrayed as scarcely conserved, repetitive in nature, quickly evolving and protein-binding competent. Among primates, the major class of centromeric DNA is the pancentromeric α-satellite, made of arrays of 171 bp monomers, repeated in a head-to-tail pattern. α-satellite sequences can either form tandem heterogeneous monomeric arrays or assemble in higher-order repeats (HORs). Gorilla centromere DNA has barely been characterized, and data are mainly based on hybridizations of human alphoid sequences. We isolated and finely characterized gorilla α-satellite sequences and revealed relevant structure and chromosomal distribution similarities with other great apes as well as gorilla-specific features, such as the uniquely octameric structure of the suprachromosomal family-2 (SF2). We demonstrated for the first time the orthologous localization of alphoid suprachromosomal families-1 and -2 (SF1 and SF2) between human and gorilla in contrast to chimpanzee centromeres. Finally, the discovery of a new 189 bp monomer type in gorilla centromeres unravels clues to the role of the centromere protein B, paving the way to solve the significance of the centromere DNA's essential repetitive nature in association with its function and the peculiar evolution of the α-satellite sequence
Structural stability of the cofactor binding site in Escherichia coli serine hydroxymethyltransferase--the role of evolutionarily conserved hydrophobic contacts.
No full textAccording to their fold, pyridoxal 5'-phosphate-dependent enzymes are grouped into five superfamilies. Fold Type I easily comprises the largest and most investigated group. The enzymes of this group have very similar 3D structures. Remarkably, the location of the cofactor in the active site, between the two domains that form a single subunit, is almost identical in all members of the group. Nonetheless, Fold Type I enzymes show very little sequence identity, raising the question as to which structural features determine the common fold. An important fold determinant appears to be the presence of three evolutionarily conserved clusters of hydrophobic contacts. A previous investigation, which used Escherichia coli serine hydroxymethyltransferase, a well characterized Fold Type I member, demonstrated the involvement of one of these clusters in the stability of the quaternary structure. The present study focuses on the role of the same cluster in the stability of the cofactor binding site. The investigation was carried out by equilibrium denaturation experiments on serine hydroxymethyltransferase forms in which the hydrophobic contact area of the cluster under study was reduced by site-directed mutagenesis. The results obtained show that the mutations clearly affected the process of pyridoxal 5'-phosphate dissociation induced by urea, reducing the stability of the cofactor binding site. We suggest that the third cluster promotes the formation of a bridging structural region that stabilizes the overall protein structure by connecting the two domains, shaping the cofactor binding site and participating in the formation of the quaternary structure
Possibili contributi dell’indagine biomolecolare in Archeologia: principi ed applicazioni
No full textRole of US elastography in the diagnosis of breast cancer
No full textIntroduction Elastography is a clinical imaging technique that assesses tissue stiffness by comparing the elasticity of a target
region with that of surrounding tissue. In breast imaging, it is employed to evaluate the stiffness of breast lesions, aiding
in their characterization. When integrated with B-mode ultrasound (US), elastography enhances the ability to distinguish
between benign, malignant, and suspicious lesions. The aim of this study was to compare the histological findings of the
breast tissue with their elastographic values and determine whether the latter, being a non-invasive technique, can help to
avoid unnecessary biopsies. Furthermore, we aim to identify cutoff values that may indicate malignancy of the lesion.
Materials and methods A retrospective study was conducted involving 40 women who underwent both shear wave elastography (SWE) and breast tissue biopsy. During the conventional B-mode ultrasound examination, SWE was also performed to
obtain quantitative elasticity measurements. The SWE parameters included the maximum stiffness value within the lesion (E),
the stiffness of the perilesional adipose tissue, and the corresponding elasticity ratio (E-ratio). Histopathological analysis of
biopsy samples was used to confirm the nature of the lesions and to serve as the reference standard for diagnostic accuracy. In
addition, B-mode ultrasound findings were compared with those obtained from SWE to assess their diagnostic performance.
Results 40 breast lesions were analyzed. E-ratio values tended to be lower in B2 lesions, consistent with benign pathology,
and higher in B4–B5 lesions, suggesting malignancy. Malignant lesions also demonstrated greater heterogeneity and significantly higher stiffness values compared to benign lesions.
Conclusions Qualitative assessment of lesion elasticity and perilesional tissue stiffness demonstrated good discriminatory
performance in differentiating breast lesions identified on ultrasound. These findings support the use of shear wave elastography as a complementary tool to conventional B-mode ultrasound in the diagnostic evaluation of breast masses
Peroxidase-like activity of Thermobifida fusca hemoglobin: The oxidation of dibenzylbutanolide
No full textThe thermostable truncated hemoglobin from the actinomyces Thermobifida fusca (Tf-trHb) displays a robust peroxidase activity, with optimum at acidic pH values, in experiments with the redox mediator ABTS. However, typical peroxidase substrates, such as phenolic or aromatic amine compounds. appear to be poor substrates for Tf-trHb. In turn, the protein is able to catalyze a unique dehydrogenation reaction of dibenzylbutanolides, suggested intermediates in the biosynthesis of podophyllotoxin, in the presence of hydrogen peroxide. Dibenzylbutanolides with a free 4 ''-hydroxyl group were thus converted into the corresponding 2,7 ''-dehydroderivatives thus setting up the basis for an efficient biotransformation of this important precursor. In particular, Tf-trHb mediated oxidation of trans-2-(4 ''-hydroxy-3 '',5 ''-dimethoxybenzyl)-3-(3',4'-methylenedioxy-7'beta-hydroxybenzyl)butanolide 1 into the corresponding benzylidene-benzoyl-gamma-butyrolactone 2 was obtained at high yield and with excellent selectivity. (C) 2009 Elsevier B.V. All rights reserved
