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32 results on '"Sica, Filomena"'

29. Exploring the unfolding mechanism of γ-glutamyltranspeptidases: The case of the thermophilic enzyme from Geobacillus thermodenitrificans

Author

Immacolata Castellano, Giuseppe Graziano, Antonello Merlino, Irene Russo Krauss, Andrea Pica, Francesco La Cara, Mosè Rossi, Filomena Sica, Pica, Andrea, RUSSO KRAUSS, Irene, Castellano, I, Rossi, M, La Cara, F, Graziano, G, Sica, Filomena, and Merlino, Antonello

Subjects
Circular dichroism, Stereochemistry, Biophysics, Cleavage (embryo), Biochemistry, Anilino Naphthalenesulfonates, Protein Structure, Secondary, Analytical Chemistry, Autocatalysis, Bacterial Proteins, Enzyme Stability, Thermal unfolding, Intermediate state, Chemical denaturation, Circular dichroism, γ-glutamyltranspeptidases, Protein stability, Transition Temperature, Molecular Biology, Protein secondary structure, Guanidine, Fluorescent Dyes, Protein Unfolding, Thermostability, chemistry.chemical_classification, Circular Dichroism, Thermophile, Geobacillus, gamma-Glutamyltransferase, Hydrogen-Ion Concentration, Amino acid, Spectrometry, Fluorescence, Enzyme, Amino Acid Substitution, chemistry, Mutagenesis, Site-Directed, Hydrophobic and Hydrophilic Interactions
Abstract

γ-glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are generally synthesized as precursor proteins, which undergo an intra-molecular autocatalytic cleavage yielding a large and a small subunit. In this study, circular dichroism and intrinsic fluorescence measurements have been used to investigate the structural features and the temperature- and guanidinium hydrochloride (GdnHCl)-induced unfolding of the mature form of the γ-GT from Geobacillus thermodenitrificans (GthGT) and that of its T353A mutant, which represents a mimic of the precursor protein. Data indicate that a) the mutant and the mature GthGT have a different secondary structure content and a slightly different exposure of hydrophobic regions, b) the thermal unfolding processes of both GthGT forms occur through a three-state model, characterized by a stable intermediate species, whereas chemical denaturations proceed through a single transition, c) both GthGT forms exhibit remarkable stability against temperature, but they do not display a strong resistance to the denaturing action of GdnHCl. These findings suggest that electrostatic interactions significantly contribute to the protein stability and that both the precursor and the mature form of GthGT assume compact and stable conformations to resist to the extreme temperatures where G. thermodenidrificans lives. Owing to its thermostability and unique catalytic properties, GthGT is an excellent candidate to be used as a glutaminase in food industry.

Published
2012

30. Crystallization of multiple forms of bovine seminal ribonuclease in the liganded and unliganded state

Author

Lelio Mazzarella, Luigi Vitagliano, Salvatore Adinolfi, Filomena Sica, Renata Piccoli, Rita Berisio, Adriana Zagari, C. De Lorenzo, Sica, F., Adinolfi, S., Berisio, R., DE LORENZO, C., Mazzarella, Lelio, Piccoli, R., Vitagliano, L., Zagari, A., Sica, Filomena, DE LORENZO, Claudia, Mazzarella, L., and Piccoli, Renata

Subjects
protein crystallization, Dimer, protein crystallization, BS-RNase, domain swapping, nucleotides, Polyethylene glycol, Condensed Matter Physics, nucleotides, law.invention, Inorganic Chemistry, chemistry.chemical_compound, Crystallography, BS-RNase, Monomer, chemistry, law, X-ray crystallography, PEG ratio, Materials Chemistry, Crystallization, domain swapping, Protein crystallization, Nucleotide, Conformational isomerism
Abstract

Bovine seminal ribonuclease (BS-RNase) is an intriguing homodimeric enzyme which exists as two conformational isomers, characterized by distinct catalytic and biological properties, referred to as M × M and M = M. Reduction of inter-chain disulfide bridges produces a stable monomeric derivative (M) which is still active. This paper reports the screening and optimization of crystallization conditions for growing single diffraction-quality crystals for the various BS-RNase forms. The crystallization trials were performed using both the vapor diffusion and microbatch methods. The M × M dimer was crystallized in the free form from polyethylene glycol (PEG) 4000 at pH 8.5 and as a complex with the substrate analog uridylyl(2′-5′)guanosine (UpG) from an unbuffered ammonium sulfate (AS) solution. These two crystal types diffract X-rays to 2.5 and 1.9 Å resolution, respectively. Two different crystal types were obtained both for the M = M dimer and for the monomeric derivative. (M = M)a crystals, grown from PEG 4000 (8% w/v) at pH 5.6, diffract X-rays to 4.0 Å. At higher PEG concentration (15% w/v) a different crystal type was obtained, (M = M)b, which showed a better diffraction limit (2.5 Å). For the monomer, type (M)a and (M)b crystals, diffracting X-rays to 2.5 Å resolution, were obtained from AS at pH 6.5 and from PEG 4000 at pH 8.5, respectively. A comparison with previously crystallized forms of the dimer M × M and its complexes with uridylyl(2′-5′)adenosine and 2′-deoxycytidylyl(3′-5′)-2′-deoxyadenosine is also presented. The three-dimensional structure analysis of (M × M) · UpG and (M = M)b is in progress. © 1999 Elsevier Science B.V. All rights reserved.

Published
1999

31. Deamidation in Proteins: The Crystal Structure of Bovine Pancreatic Ribonuclease with an Isoaspartyl Residue at Position 67

Author

Luigi Vitagliano, Filomena Sica, Adriana Zagari, Giosué Sorrentino, Sante Capasso, Luciana Esposito, Lelio Mazzarella, A. Di Donato, S., Capasso, DI DONATO, Alberto, L., Esposito, Sica, Filomena, G., Sorrentino, L., Vitagliano, A., Zagari, and Mazzarella, L.

Subjects
biology, Protein Conformation, Ribonuclease, Pancreatic, Crystallography, X-Ray, Bovine pancreatic ribonuclease, chemistry.chemical_compound, Residue (chemistry), Crystallography, Protein structure, chemistry, Structural Biology, Hydrolase, biology.protein, Animals, Cattle, Ribonuclease, Asparagine, Crystallization, Deamidation, Molecular Biology, Derivative (chemistry)
Abstract

The non-enzymatic deamidation of asparagine residues in proteins is a widely occurring reaction, both in vivo and in vitro. Although the importance of this process is commonly recognised, only little structural information is available on it. In order to evaluate the structural effects of this reaction in proteins, we have determined the crystal structure of a ribonuclease A derivative in which asparagine 67 has been replaced by an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. The overall structure of the model, refined to a crystallographic R-factor of 0.159 at a resolution of 1.9 A, is very similar to that of the native protein, but considerable deviations are observed in the region delimited by the disulphide bridge 65-72. In particular, the insertion of an extra methylene group in the main chain at residue 67 breaks up the hydrogen bond network that makes this region rather rigid in ribonuclease A. On the basis of the structure observed, some of the slightly but significantly different properties of this deamidated derivative, with respect to the native enzyme, can be explained.

Published
1996

32. Crystallization and Preliminary X-ray Analysis of an NAD+ -dependent Alcohol Dehydrogenase from the Extreme Thermophilic Archaebacterium Sulfolobus solfataricus

Author

Andrew M. Hemmings, Carlo A. Raia, Domenico Demasi, Mosè Rossi, Lelio Mazzarella, Filomena Sica, Laurence H. Pearl, Sabato D' Auria, L. H., Pearl, D., Demasi, A. M., Hemming, Sica, Filomena, L., Mazzarella, C. A., Raia, S., Dauria, and M., Rossi

Subjects
biology, ved/biology, Dimer, Thermophile, Sulfolobus solfataricus, ved/biology.organism_classification_rank.species, Alcohol Dehydrogenase, Polyethylene glycol, NAD, Sulfolobus, law.invention, chemistry.chemical_compound, Crystallography, Monomer, X-Ray Diffraction, chemistry, Structural Biology, law, biology.protein, NAD+ kinase, Crystallization, Molecular Biology, Alcohol dehydrogenase
Abstract

An NAD + -dependent alcohol dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus has been crystallized in the holo-enzyme and apo-enzyme forms. Crystals of the holo-enzyme grow from 2-methyl-2,4-pentanediol at pH 8·4 with the addition of NADH and at pH 7·0 with the addition of NADH and dimethyl sulphoxide. Crystals of the apo-enzyme grow at pH 6·3 from a mixture of polyethylene glycol 4000 and propan-2-ol. The holo-enzyme crystallizes in C 2 with a dimer in the asymmetric unit, however the crystals are twinned and unsuitable for data collection. The apo-enzyme crystallizes in I 4 1 22 ( a = 126·82 A, b = 118·95 A) with a monomer in the asymmetric unit, and the single crystals diffract to 2·8 A.

Published
1993

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