Insights into the Specificity of Thioredoxin Reductase-Thioredoxin Interactions. A Structural and Functional Investigation of the Yeast Thioredoxin System

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural depth profile and nanoscale piezoelectric properties of randomly oriented Pb(Zr0.50Ti0.50)O-3 thin films

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic variability of porcine parvovirus isolates revealed by analysis of partial sequences of the structural coding gene VP2

The 3'-terminal 853 nt (and the putative 283 aa) sequence of the VP2-encoding gene from 29 field strains of porcine parvovirus (PPV) were determined and compared both to each other and with other published sequences. Sequences were examined using maximum-parsimony and statistical analyses for nucleotide diversity and sequence variability. Among the nucleotide sequences of the PPV field strains, 26 polymorphic sites were encountered; 22 polymorphic sites were detected in the putative amino acid sequence. Mapping polymorphic sites of protein data onto the three-dimensional (3D) structure of PPV VP2 revealed that almost all substitutions were located on the external surface of the viral capsid. Mapping amino acid substitutions to the alignment between PPV VP2 sequences and the 3D structure of canine parvovirus (CPV) capsid, many PPV substitutions were observed to map to regions of recognized antigenicity and/or to contain phenotypically important residues for CPV and other parvoviruses. In spite of the high sequence similarity, genetic analysis has shown the existence of at least two virus lineages among the samples. In conclusion, these results highlight the need for close surveillance on PPV genetic drift, with an assessment of its potential ability to modify the antigenic make-up of the virus.

Relationship between global structural parameters and Enzyme Commission hierarchy: Implications for function prediction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural basis for inhibition of cyclin-dependent kinase 9 by flavopiridol

Flavopiridol has been shown to potently inhibit CDK1 and 2 (cyclin-dependent kinases 1 and 2) and most recently it has been found that it also inhibits CDK9. The complex CDK9-cyclin T1 controls the elongation phase of transcription by RNA polymerase II. The present work describes a molecular model for the binary complex CDK9-flavopiridol. This structural model indicates that the inhibitor strongly binds to the ATP-binding pocket of CDK9 and the structural comparison of the complex CDK2-flavopiridol correlates the structural differences with differences in inhibition of these CDKs by flavopiridol. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known leading structures such as flavones and adenine derivatives. © 2002 Elsevier Science (USA). All rights reserved.

A new firefly luciferase with bimodal spectrum: Identification of structural determinants of spectral pH-sensitivity in firefly luciferases

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases. © 2005 American Society for Photobiology.

Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/β)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients. © 2013 Elsevier Ltd.