472 results on '"Sica, Filomena"'
Search Results
202. Identification of Aminosuccinyl Residues In Peptides By 2nd-derivative Ultraviolet Spectrometry
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S. CAPASSO, A. ZAGARI, SICA, FILOMENA, S., Capasso, Sica, Filomena, and A., Zagari
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- 1987
203. Identification of aminosuccinyl residues in peptides by second-derivative ultraviolet spectrometry
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CAPASSO SANTE, ZAGARI A.D.R.I.A.N.A., SICA, FILOMENA, Capasso, Sante, Sica, Filomena, and Zagari, A. D. R. I. A. N. A.
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- 1987
204. A potential allosteric subsite generated by domain swapping in bovine seminal ribonuclease11Edited by A. R. Fersht
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Vitagliano, Luigi, Adinolfi, Salvatore, Sica, Filomena, Merlino, Antonello, Zagari, Adriana, and Mazzarella, Lelio
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Bovine seminal ribonuclease (BS-RNase) is a peculiar member of the pancreatic-like ribonuclease superfamily endowed with unique biological functions. It has been shown that native BS-RNase is a mixture of two distinct dimeric forms. The most abundant form is characterised by the swapping of the N-terminal helix. Kinetic studies have shown that this dimer is allosterically regulated, whereas the minor component, in which no swapping occurs, exhibits typical Michaelian kinetics. In order to correlate the catalytic properties with the structural features of BS-RNase, we have determined the crystal structure of the BS-RNase swapping dimer complexed with uridylyl(2′-5′)guanosine. The structure of the complex was refined to an Rvalue of 0.189 at 1.9 Å resolution. Surprisingly, the enzyme binds four dinucleotide molecules, all in a non-productive way. In the two active sites, the guanine base is located in the subsite that is specific for pyrimidines. This unusual binding has been observed also in complexes of RNase A with guanine-containing nucleotides (retro-binding). One of the two additional dinucleotide molecules bound to the enzyme is located on the surface of the protein in a pocket generated by crystal packing; the second was found in a cavity at the interface between the two subunits of the swapping dimer. There are indications that the interface site plays a role in the allosteric regulation exhibited by BS-RNase. This finding suggests that domain swapping may not merely be a mechanism that proteins adopt for the transition from a monomeric to oligomeric state but can be used to achieve modulations in catalytic function.
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- 1999
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205. Raman imaging and tweezers of red blood cells from patients affected by anemia S, C and C+S
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Balsamo, A., Pully, V., Giulia Rusciano, Sica, Filomena, Antonello Merlino, Michela Grosso, Sasso, Antonio, Mazzarella, Lelio, Otto, C., Alessandro Vergara, A., Balsamo, V., Pully, Rusciano, Giulia, Sica, Filomena, Merlino, Antonello, Grosso, Michela, Sasso, Antonio, Mazzarella, Lelio, C., Otto, and Vergara, Alessandro
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cellula singola ,emoglobina ,Raman - Abstract
Raman imaging visualizes without the need for labeling the molecular composition of sub-cellular compartments [1]. Resonance Raman spectroscopy elucidates the structural characteristics of heme domains in hemoproteins [2]. Optical Tweezers (OT) allows a rapid identification of single-cell mechanical properites [3]. Haemoglobinopathies are inherited disorders defined as qualitative or quantitative deficiency of haemoglobin (Hb) in red blood cells (RBCs). Herein we analyze anemic RBCs with two human Hb variants, HbS and HbC (Glu6Val and Glu6Lys, respectively). A comparative study in a family (mother, father and daughter, respectively heterozygous for HbS, HbC and Hb C+S) shows that different Raman images and mechanical properties of healthy and anemic RBCs could help to evaluate the expression ratios between HbS and HbC, and correlate these data with varied clinical conditions. 1. van Manen H.J., et al., Proc. Natl. Acad. Sci. 2005, 102, 10159. 2. Vitagliano L., et al., J. Am. Chem. Soc. 2008, 130, 10527. 3. De Luca A.C., et al., Opt. Exp. 2008, 16(11), 7943.
206. Relazione tra pH, struttura ed affinità per l’ossigeno nell’emoglobina con effetto Root da Trematomus bernacchii: un approccio combinato teorico-sperimentale
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Antonello Merlino, Alessandro Vergara, Sica, Filomena, Balsamo, A., Verde, C., Boechi, L., Marti’, M. A., Estrin, D. A., Ronda, L., Mozzarelli, A., Mazzarella, Lelio, Merlino, Antonello, Vergara, Alessandro, Sica, Filomena, A., Balsamo, C., Verde, L., Boechi, M. A., Marti’, D. A., Estrin, L., Ronda, A., Mozzarelli, and Mazzarella, Lelio
207. Structure, stability and dynamics of a mesophilic-psychrophilc pair
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Russo Krauss, Irene, Antonello Merlino, Immacolata Castellano, Vendittis, Emmanuele, Conte, M., Alessandro Vergara, Sica, Filomena, RUSSO KRAUSS, Irene, Merlino, Antonello, Castellano, Immacolata, DE VENDITTIS, Emmanuele, M., Conte, Vergara, Alessandro, and Sica, Filomena
208. Identification of aminosuccinyl residues in peptides by second-derivative ultraviolet spectrometry
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Capasso, Sante, primary, Sica, Filomena, additional, and Zagari, Adriana, additional
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- 1987
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209. Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases
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Rosanna Culurciello, Andrea Bosso, Romualdo Troisi, Valentina Barrella, Ilaria Di Nardo, Margherita Borriello, Rosa Gaglione, Valeria Pistorio, Serena Aceto, Valeria Cafaro, Eugenio Notomista, Filomena Sica, Angela Arciello, Elio Pizzo, Culurciello, Rosanna, Bosso, Andrea, Troisi, Romualdo, Barrella, Valentina, Di Nardo, Ilaria, Borriello, Margherita, Gaglione, Rosa, Pistorio, Valeria, Aceto, Serena, Cafaro, Valeria, Notomista, Eugenio, Sica, Filomena, Arciello, Angela, and Pizzo, Elio
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Keratinocytes ,oxidative stre ,stress induced RNases ,tiRNA ,stress granules ,skin cells ,oxidative stress ,Organic Chemistry ,stress induced RNase ,Ribonuclease, Pancreatic ,General Medicine ,stress granule ,Catalysis ,Computer Science Applications ,Inorganic Chemistry ,Oxidative Stress ,Ribonucleases ,RNA, Transfer ,Humans ,Physical and Theoretical Chemistry ,Molecular Biology ,Keratinocyte ,Spectroscopy ,Human ,skin cell - Abstract
Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.
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- 2022
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210. A terminal functionalization strategy reveals unusual binding abilities of anti-thrombin anticoagulant aptamers
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Romualdo Troisi, Claudia Riccardi, Kévan Pérez de Carvasal, Michael Smietana, François Morvan, Pompea Del Vecchio, Daniela Montesarchio, Filomena Sica, Troisi, Romualdo, Riccardi, Claudia, Pérez de Carvasal, Kévan, Smietana, Michael, Morvan, Françoi, Del Vecchio, Pompea, Montesarchio, Daniela, and Sica, Filomena
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modified oligonucleotide ,MT: Oligonucleotides: Therapies and application ,crystal structure ,anticoagulant ,Drug Discovery ,aptamer ,Molecular Medicine ,coagulation factor - Abstract
Despite their unquestionable properties, oligonucleotide aptamers display some drawbacks that continue to hinder their applications. Several strategies have been undertaken to overcome these weaknesses, using thrombin binding aptamers as proof-of-concept. In particular, the functionalization of a thrombin exosite I binding aptamer (TBA) with aromatic moieties, e.g., naphthalene dimides (N) and dialkoxynaphthalenes (D), attached at the 5' and 3' ends, respectively, proved to be highly promising. To obtain a molecular view of the effects of these modifications on aptamers, we performed a crystallographic analysis of one of these engineered oligonucleotides (TBA-NNp/DDp) in complex with thrombin. Surprisingly, three of the four examined crystallographic structures are ternary complexes in which thrombin binds a TBA-NNp/DDp molecule at exosite II as well as at exosite I, highlighting the ability of this aptamer, differently from unmodified TBA, to also recognize a localized region of exosite II. This novel ability is strictly related to the solvophobic behavior of the terminal modifications. Studies were also performed in solution to examine the properties of TBA-NNp/DDp in a crystal-free environment. The present results throw new light on the importance of appendages inducing a pseudo-cyclic charge-transfer structure in nucleic acid-based ligands to improve the interactions with proteins, thus considerably widening their potentialities.
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- 2022
211. Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands
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Luigi Vitagliano, Romualdo Troisi, Ida Autiero, Filomena Sica, Nicole Balasco, Troisi, Romualdo, Balasco, Nicole, Autiero, Ida, Vitagliano, Luigi, and Sica, Filomena
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Models, Molecular ,natural inhibitor ,QH301-705.5 ,Aptamer ,exosite ,Allosteric regulation ,Protein Data Bank (RCSB PDB) ,Computational biology ,Review ,Ligands ,Catalysis ,Hemostatics ,Substrate Specificity ,Inorganic Chemistry ,Thrombin ,functional partner ,synthetic compound ,medicine ,Animals ,Humans ,structure ,Physical and Theoretical Chemistry ,Biology (General) ,Molecular Biology ,QD1-999 ,Spectroscopy ,chemistry.chemical_classification ,dynamic ,Binding Sites ,allostery ,Biomolecule ,Organic Chemistry ,aptamer ,General Medicine ,computer.file_format ,Aptamers, Nucleotide ,Protein Data Bank ,inter-exosite communication ,Computer Science Applications ,Chemistry ,chemistry ,Functional activity ,computer ,medicine.drug ,Discovery and development of direct thrombin inhibitors ,Protein Binding - Abstract
Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.
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- 2021
212. Aptamers: Functional-Structural Studies and Biomedical Applications
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Romualdo Troisi, Filomena Sica, Troisi, Romualdo, and Sica, Filomena
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Inorganic Chemistry ,SELEX Aptamer Technique ,Organic Chemistry ,DNA ,General Medicine ,Aptamers, Nucleotide ,Physical and Theoretical Chemistry ,Molecular Biology ,Spectroscopy ,Catalysis ,Computer Science Applications - Abstract
Aptamers are synthetic molecules of different natures (mostly, DNA or RNA) that recognize a target molecule with high affinity and specificity [...]
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- 2022
213. Structural and functional analysis of the simultaneous binding of two duplex/quadruplex aptamers to human α-thrombin
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Luigi Vitagliano, Andreas Santamaria, Romualdo Troisi, Filomena Sica, Nicole Balasco, Troisi, Romualdo, Balasco, Nicole, Santamaria, Andrea, Vitagliano, Luigi, and Sica, Filomena
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Time Factors ,Aptamer ,Anti-thrombin aptamer ,02 engineering and technology ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Biochemistry ,03 medical and health sciences ,Molecular dynamics ,Thrombin ,Structural Biology ,medicine ,Humans ,Molecular Biology ,Ternary complex ,Blood Coagulation ,030304 developmental biology ,0303 health sciences ,Functional analysis ,Chemistry ,Anticoagulants ,Fibrinogen ,General Medicine ,Aptamers, Nucleotide ,021001 nanoscience & nanotechnology ,Thrombin modulation ,α thrombin ,Inter-exosite communication ,Duplex (building) ,Biophysics ,0210 nano-technology ,medicine.drug ,Discovery and development of direct thrombin inhibitors ,Protein Binding - Abstract
The long-range communication between the two exosites of human α-thrombin (thrombin) tightly modulates the protein-effector interactions. Duplex/quadruplex aptamers represent an emerging class of very effective binders of thrombin. Among them, NU172 and HD22 aptamers are at the forefront of exosite I and II recognition, respectively. The present study investigates the simultaneous binding of these two aptamers by combining a structural and dynamics approach. The crystal structure of the ternary complex formed by the thrombin with NU172 and HD22_27mer provides a detailed view of the simultaneous binding of these aptamers to the protein, inspiring the design of novel bivalent thrombin inhibitors. The crystal structure represents the starting model for molecular dynamics studies, which point out the cooperation between the binding at the two exosites. In particular, the binding of an aptamer to its exosite reduces the intrinsic flexibility of the other exosite, that preferentially assumes conformations similar to those observed in the bound state, suggesting a predisposition to interact with the other aptamer. This behaviour is reflected in a significant increase of the anticoagulant activity of NU172 when the inactive HD22_27mer is bound to exosite II, providing a clear evidence of the synergic action of the two aptamers.
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- 2021
214. Expanding the recognition interface of the thrombin-binding aptamer HD1 through modification of residues T3 and T12
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Filomena Sica, Natalia A. Kolganova, Edward N. Timofeev, Romualdo Troisi, Igor P. Smirnov, Smirnov, Igor, Kolganova, Natalia, Troisi, Romualdo, Sica, Filomena, and Timofeev, Edward
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0301 basic medicine ,anticoagulants ,crystal structure ,Pyrimidine ,post-SELEX modifications ,Aptamer ,Mutant ,G-quadruplex ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Thrombin ,binding affinity ,Drug Discovery ,medicine ,Side chain ,Nucleotide ,chemistry.chemical_classification ,Thrombin-binding aptamer ,post-SELEX modifications thrombin-binding aptamer anticoagulants crystal structure G-quadruplex binding affinity ,lcsh:RM1-950 ,lcsh:Therapeutics. Pharmacology ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Biophysics ,Molecular Medicine ,Original Article ,thrombin-binding aptamer ,medicine.drug - Abstract
Post-SELEX modification of DNA aptamers is an established strategy to improve their affinity or inhibitory characteristics. In this study, we examined the possibility of increasing the recognition interface between the thrombin-binding aptamer HD1 (TBA) and thrombin by adding a chemically modified side chain to selected nucleotide residues. A panel of 22 TBA variants with N3-modified residues T3 and T12 was prepared by a two-step modification procedure. Aptamers were characterized by a combination of biophysical and biochemical methods. We identified mutants with enhanced affinity and improved anticoagulant activity. The crystal structures of thrombin complexes with three selected modified variants revealed that the modified pyrimidine base invariably allocates in proximity to thrombin residues Tyr76 and Ile82 due to the directing role of the unmodified TT loop. The modifications induced an increase in the contact areas between thrombin and the modified TBAs. Comparative analysis of the structural, biochemical, and biophysical data suggests that the non-equivalent binding modes of the mutants with thrombin in the T3- and T12-modified series account for the observed systematic differences in their affinity characteristics. In this study, we show that extending the recognition surface between the protein and modified aptamers is a promising approach that may improve characteristics of aptamer ligands., Graphical Abstract, Smirnov et al. describe a post-SELEX approach for the thrombin aptamer HD1 that consists in expanding the aptamer-protein interface through attachment of non-natural side chains at specific aptamer residues. The authors report synthesis and characterization of 22 aptamer mutants and present crystal structures of thrombin complexes with three selected variants.
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- 2021
215. New structural information expands the understanding of the thrombin modulation with oligonucleotide aptamers
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Filomena Sica, Associazione Italiana Cristallografia, and Sica, Filomena
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Thrombin, aptamers, allostery - Abstract
During the last decades, thrombin binding aptamer (TBA) was one of the most extensively studied oligonucleotide G-quadruplex aptamers [1] and, despite the advent of new anti-thrombin aptamers [2], the search for mutations that are able to improve TBA properties is still ongoing [3]. In this context, an extensive analysis of twenty-two TBA variants with N3-modified residues Thy3 and Thy12, two key residues of the two TT loops that guide the binding with thrombin, was performed in the course of a collaborative research with Engelhardt Institute of Molecular Biology (Russian Academy of Sciences, Russia). Among them, some mutants with enhanced affinity and improved anticoagulant activity were identified (data to be published). Here, we present the crystal structures of the complexes between thrombin and three of these TBA variants, containing a modification in the Thy3 nucleobase. The data revealed that the modified pyrimidine base invariably allocates in proximity to thrombin residues Tyr76 and Ile82 due to the directing role of the unmodified TT loop. Moreover, the modifications were found to induce an increase in the contact areas between thrombin and the modified TBAs by a slight conformation adaptation of the aptamer to the thrombin binding site. Details will be presented in the poster.
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- 2020
216. Several structural motifs cooperate in determining the highly effective anti-thrombin activity of NU172 aptamer
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Valeria Napolitano, Romualdo Troisi, I. Russo Krauss, V. A. Spiridonova, Filomena Sica, Troisi, Romualdo, Napolitano, Valeria, Spiridonova, Vera, RUSSO KRAUSS, Irene, and Sica, Filomena
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Aptamer ,Amino Acid Motifs ,Oligonucleotides ,Plasma protein binding ,030204 cardiovascular system & hematology ,Biology ,Crystallography, X-Ray ,Ligands ,G-quadruplex ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Thrombin ,Fibrinolytic Agents ,Structural Biology ,Hydrolase ,Genetics ,medicine ,Humans ,Structural motif ,Circular Dichroism ,Anticoagulants ,Fibrinogen ,Aptamers, Nucleotide ,G-Quadruplexes ,030104 developmental biology ,Duplex (building) ,Mutation ,Biophysics ,Protein Binding ,medicine.drug - Abstract
Despite aptamers are very promising alternative to antibodies, very few of them are under clinical trials or are used as drugs. Among them, NU172 is currently in Phase II as anticoagulant in heart disease treatments. It inhibits thrombin activity much more effectively than TBA, the best-known thrombin binding aptamer. The crystal structure of thrombin-NU172 complex reveals a bimodular duplex/quadruplex architecture for the aptamer, which binds thrombin exosite I through a highly complementary surface involving all three loops of the G-quadruplex module. Although the duplex domain does not interact directly with thrombin, the features of the duplex/quadruplex junction and the solution data on two newly designed NU172 mutants indicate that the duplex moiety is important for the optimization of the protein-ligand interaction and for the inhibition of the enzyme activity. Our work discloses the structural features determining the inhibition of thrombin by NU172 and put the basis for the design of mutants with improved properties.
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- 2018
217. Molecular dynamics simulations shed light on the cooperative mechanisms generated by the simultaneous binding of aptamers at the two exosites of thrombin'
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Filomena Sica, Romualdo Troisi, Nicole Balasco, Croatian Association of Crystallographers, Sica, Filomena, Troisi, Romualdo, and Balasco, Nicole
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Molecular dynamics simulations shed light on the cooperative mechanisms generated by the simultaneous binding of aptamers at the two exosites of thrombin Human α-thrombin is a trypsin-like serine protease endowed with the unique ability to convert soluble fibrinogen in insoluble fibrin clot. In addition to the active site, this enzyme owns two electropositive regions, exosite I and II, located at opposite sides of its globular shape [1]. The narrow substrate specificity of thrombin and its ability to change function are regulated by the exosite binding to different cofactors and modulators [2]. A special class of thrombin exosite synthetic ligands is represented by G-quadruplex anticoagulant aptamers, which are short single stranded DNA or RNA oligonucleotides that bind their targets with very high affinity and specificity [3]. The minimal 15mer DNA aptamer, named TBA, is the first and the most studied anti-thrombin aptamer. Based on several X-ray studies, this aptamer was established to bind the fibrinogen-binding site of thrombin (exosite I) by a pincer-like recognition mechanism involving the two TT loops [4]. The addition of a duplex motif to the G-quadruplex module has produced a new generation of aptamers with higher affinity against thrombin compared to TBA [5]. Among them, an aptamer, named HD22_27mer, recognizes exosite II with both quadruplex and duplex domains [6].In the last years, great attention has been paid to the study of the effects of the simultaneous binding of two ligands on the two exosites of thrombin. Biochemical studies have suggested that thrombin is an allosterically modulated enzyme: an interplay between its two exosites as well as between the exosites and the active site has been highlighted [7]. Furthermore, the crystallographic structures of two thrombin ternary complexes, in which exosite II is bound to HD22_27mer and exosite I interacts with TBA-like aptamers, were solved and gave structural information on the effects of the simultaneous binding of two aptamers to thrombin exosites [8]. Here we present the results of an extensive molecular dynamics study performed on free thrombin and on its binary and ternary complexes with TBA and HD22_27mer in the absence of the PPACK inhibitor that is covalently bound to the protein active site in all the crystallographic α-thrombin models. This study revealed that, in the absence of any influence of the crystal packing, an inter-exosite cross-talk and an active site-exosite communication may occur in these systems. Details will be discussed at the Meeting.[1] E. Di Cera, J Thromb Haemost., 5, (2007), 196-202.[2] S. Krishnaswamy, J Thromb Haemost., 3, (2005), 54-67.[3] A.D. Keefe, S. Pai, A. Ellington, Nat Rev Drug Discov., 9, (2010), 537-550.[4] I. Russo Krauss, A. Merlino, A. Randazzo, E. Novellino, L. Mazzarella, F. Sica, Nucleic Acids Res., 40, (2012), 8119-8128.[5] I. Russo Krauss, V. Napolitano, L. Petraccone, R. Troisi, V. Spiridonova, C.A. Mattia, F. Sica, Int J Biol Macromol., 107, (2018), 1697-1705.[6] I. Russo Krauss, A. Pica, A. Merlino, L. Mazzarella, F. Sica, Acta Cryst. D, 69, (2013), 2403-2411.[7] N.S. Petrera, A.R. Stafford, B.A. Leslie, C.A. Kretz, J.C. Fredenburgh, J.I. Weitz, J Biol Chem., 284, (2009), 25620-25629.[8] A. Pica, I. Russo Krauss, V. Parente, H. Tateishi-Karimata, S. Nagatoishi, K. Tsumoto, N. Sugimoto, F. Sica, Nucleic Acids Res., 45, (2017), 461-469
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- 2019
218. Communication between thrombin exosites: structural effects of the simultaneous binding of two bimodular aptamers
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R. Troisi, SANTAMARIA, ANDREAS, N. Balasco, Filomena Sica, Associazione Italiana Cristallografia, Troisi, R., Santamaria, Andrea, Balasco, N., and Sica, Filomena
- Abstract
Human α-thrombin is a trypsin-like serine protease playing a pivotal role in the haemostatic pathway. Apart from the active site, thrombin has two electropositive patches on its surface, named exosites I and II, which determine the biological properties of the enzyme through the binding of substrates and cofactors [1,2]. The two exosites also represent the anchoring sites for aptamers, which are short single stranded DNA or RNA oligonucleotides that bind their targets with very high affinity and specificity, by adopting defined and stable 3D structures [3,4]. New generation anti-thrombin aptamers, which adopt a mixed duplex/quadruplex structure and possess improved binding or anticoagulant properties, are currently under study [5]. Among them, NU172, the only thrombin binding aptamer in advanced clinical trial, recognizes exosite I with its quadruplex domain [6]. On the other hand, HD22_27mer oligonucleotide binds exosite II with both quadruplex and duplex domains [7]. Both these bimodular aptamers show high affinity toward their target. Recently, great attention has been paid to the study of the effects of the simultaneous binding of two ligands on both thrombin exosites [8-11]. In particular, an efficient modulation of thrombin activity could be obtained by the combined use of NU172 and HD22_27mer. Here we present the crystal structure of the ternary complex, in which thrombin is sandwiched between NU172 at exosite I and HD22_27mer at exosite II. Even though the crystalline packing reduces the conformational mobility of the two aptamers, the comparison of the present model with those of the relative binary complexes has revealed small but significant clues of an inter-exosites cross-talk. To evaluate the actual influence of the crystal packing on the long-range communication between the two exosites, a molecular dynamics study of the ternary complex is in progress. Details will be discussed at the Meeting. [1] E. Di Cera J Thromb Haemost. 2007, 5, 196. [2] S. Krishnaswamy J Thromb Haemost. 2005, 3, 54. [3] A.D. Keefe, S. Pai, A. Ellington Nat Rev Drug Discov. 2010, 9, 537. [4] I. Russo Krauss, A. Merlino, A. Randazzo, E. Novellino, L. Mazzarella, F. Sica Nucleic Acids Res. 2012, 40, 8119. [5] I. Russo Krauss, V. Napolitano, L. Petraccone, R. Troisi, V. Spiridonova, C.A. Mattia, F. Sica Int J Biol Macromol. 2018, 107, 1697. [6] R. Troisi, V. Napolitano, V. Spiridonova, I. Russo Krauss, F. Sica Nucleic Acids Res. 2018, 46, 12177. [7] I. Russo Krauss, A. Pica, A. Merlino, L. Mazzarella, F. Sica Acta Cryst. D 2013, 69, 2403. [8] N.S. Petrera, A.R. Stafford, B.A. Leslie, C.A. Kretz, J.C. Fredenburgh, J.I. Weitz J Biol Chem. 2009, 284, 25620. [9] X. Feng, C. Yu, F. Feng, P. Lu, Y. Chai, Q. Li, D. Zhang, X. Wang, L. Yao Chemistry 2019, 25, 2978. [10] K. Derszniak, K. Przyborowski, K. Matyjaszczyk, M. Moorlag, B. de Laat, M. Nowakowska, S. Chlopicki Front Pharmacol. 2019, 10, 68. [11] A. Pica, I. Russo Krauss, V. Parente, H. Tateishi-Karimata, S. Nagatoishi, K. Tsumoto, N. Sugimoto, F. Sica Nucleic Acids Res., 2017, 45, 461.
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- 2019
219. Duplex/quadruplex oligonucleotides: Role of the duplex domain in the stabilization of a new generation of highly effective anti-thrombin aptamers
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Carlo Andrea Mattia, Valeria Napolitano, Luigi Petraccone, V. A. Spiridonova, Romualdo Troisi, Irene Russo Krauss, Filomena Sica, Russo Krauss, Irene, Napolitano, Valeria, Petraccone, Luigi, Troisi, Romualdo, Spiridonova, Vera, Mattia, Carlo Andrea, and Sica, Filomena
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0301 basic medicine ,Circular dichroism ,Aptamer ,Duplex/quadruplex ,Ultraviolet Rays ,Oligonucleotides ,Monovalent cation ,010402 general chemistry ,G-quadruplex ,Nucleic Acid Denaturation ,01 natural sciences ,Biochemistry ,TBA ,Antithrombins ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,heterocyclic compounds ,Molecular Biology ,Coagulation ,Base Sequence ,Calorimetry, Differential Scanning ,Oligonucleotide ,Circular Dichroism ,Bimodular aptamer ,Structure-function relationship ,Thrombin ,Temperature ,DNA ,General Medicine ,Aptamers, Nucleotide ,0104 chemical sciences ,G-Quadruplexes ,030104 developmental biology ,chemistry ,Structural biology ,Duplex (building) ,Biophysics ,Anti-thrombin aptamers - Abstract
Recently, mixed duplex/quadruplex oligonucleotides have attracted great interest for use as biomedical aptamers. In the case of anti-thrombin aptamers, the addition of duplex-forming sequences to a G-quadruplex module identical or very similar to the best-known G-quadruplex of the Thrombin Binding Aptamer (HD1) results in new or improved biological properties, such as higher activity or different recognition properties with respect to HD1. Remarkably, this bimodular fold was hypothesized, based on its sequence, for the only anti-thrombin aptamer in advanced clinical trial, NU172. Whereas cation modulation of G-quadruplex conformation and stability is well characterized, only few data from similar analysis on duplex/quadruplex oligonucleotides exist. Here we have performed a characterization of structure and stability of four different duplex/quadruplex anti-thrombin aptamers, including NU172, in the presence of different cations and in physiological-mimicking conditions in comparison to HD1, by means of spectroscopic techniques (UV and circular dichroism) and differential scanning calorimetry. Our data show a strong reciprocal influence of each domain on the stability of the other and in particular suggest a stabilizing effect of the duplex region in the presence of solutions mimicking the physiological conditions, strengthening the idea that bimodular aptamers present better therapeutic potentialities than those containing a single G-quadruplex domain.
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- 2017
220. High yield purification and first structural characterization of the full-length bacterial toxin CNF1
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Maria Luisa Tutino, Marco Caterino, Alessia Fabbri, Filomena Sica, Andrea Colarusso, Alessandro Vergara, Carla Fiorentini, Ermenegilda Parrilli, Colarusso, Andrea, Caterino, Marco, Fabbri, Alessia, Fiorentini, Carla, Vergara, Alessandro, Sica, Filomena, Parrilli, Ermenegilda, and Tutino, Maria Luisa
- Subjects
0301 basic medicine ,Circular dichroism ,RHOA ,Size-exclusion chromatography ,Bacterial Toxins ,medicine.disease_cause ,Secondary structure analysi ,Chromatography, Affinity ,law.invention ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,law ,Protein purification ,medicine ,Escherichia coli ,Humans ,Protein secondary structure ,biology ,Toxin ,Chemistry ,Escherichia coli Proteins ,Recombinant protein production ,CNF1 ,Recombinant Proteins ,030104 developmental biology ,Biochemistry ,biology.protein ,Recombinant DNA ,rhoA GTP-Binding Protein ,030217 neurology & neurosurgery ,Biotechnology - Abstract
The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150-159, 2018.
- Published
- 2017
221. Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers
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Andrea Pica, Valeria Parente, Satoru Nagatoishi, Naoki Sugimoto, Kouhei Tsumoto, Hisae Tateishi-Karimata, Irene Russo Krauss, Filomena Sica, Pica, Andrea, RUSSO KRAUSS, Irene, Parente, Valeria, Tateishi Karimata, Hisae, Nagatoishi, Satoru, Tsumoto, Kouhei, Sugimoto, Naoki, and Sica, Filomena
- Subjects
0301 basic medicine ,Models, Molecular ,Aptamer ,Plasma protein binding ,DNA Aptamers ,Biology ,Crystallography, X-Ray ,03 medical and health sciences ,Thrombin ,Structural Biology ,Genetics ,medicine ,Molecule ,Humans ,Human Thrombin ,Protein Interaction Domains and Motifs ,Binding site ,Binding Sites ,Aptamers, Nucleotide ,Molecular biology ,Combinatorial chemistry ,030104 developmental biology ,Nucleic Acid Conformation ,Thermodynamics ,Ternary operation ,medicine.drug ,Protein Binding - Abstract
Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.
- Published
- 2017
222. A regular thymine tetrad and a peculiar supramolecular assembly in the first crystal structure of an all-LNA G-quadruplex
- Author
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Antonello Merlino, Filomena Sica, Carlo Andrea Mattia, Gary N. Parkinson, Antonio Randazzo, Irene Russo Krauss, Lelio Mazzarella, Ettore Novellino, RUSSO KRAUSS, Irene, Parkinson, Gn, Merlino, Antonello, Mattia, Ca, Randazzo, Antonio, Novellino, Ettore, Mazzarella, L, and Sica, Filomena
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Models, Molecular ,G-quadruplex ,Oligonucleotides ,Sequence (biology) ,General Medicine ,Methylene bridge ,Crystal structure ,Crystallography, X-Ray ,Thymine ,Supramolecular assembly ,G-Quadruplexes ,chemistry.chemical_compound ,Crystallography ,chemistry ,Biochemistry ,Structural Biology ,Thermodynamics ,Locked nucleic acid ,Tetrad - Abstract
Locked nucleic acids (LNAs) are formed by bicyclic ribonucleotides where the O2′ and C4′ atoms are linked through a methylene bridge and the sugar is blocked in a 3′-endoconformation. They represent a promising tool for therapeutic and diagnostic applications and are characterized by higher thermal stability and nuclease resistance with respect to their natural counterparts. However, structural descriptions of LNA-containing quadruplexes are rather limited, since few NMR models have been reported in the literature. Here, the first crystallographically derived model of an all-LNA-substituted quadruplex-forming sequence 5′-TGGGT-3′ is presented refined at 1.7 Å resolution. This high-resolution crystallographic analysis reveals a regular parallel G-quadruplex arrangement terminating in a well defined thymine tetrad at the 3′-end. The detailed picture of the hydration pattern reveals LNA-specific features in the solvent distribution. Interestingly, two closely packed quadruplexes are present in the asymmetric unit. They face one another with their 3′-ends giving rise to a compact higher-order structure. This new assembly suggests a possible way in which sequential quadruplexes can be disposed in the crowded cell environment. Furthermore, as the formation of ordered structures by molecular self-assembly is an effective strategy to obtain nanostructures, this study could open the way to the design of a new class of LNA-based building blocks for nanotechnology.
- Published
- 2014
223. The multiple forms of bovine seminal ribonuclease: Structure and stability of a C-terminal swapped dimer
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Delia Picone, Andrea Pica, Filomena Sica, Antonello Merlino, Irene Russo Krauss, Carmine Ercole, Sica, Filomena, Pica, Andrea, Merlino, Antonello, RUSSO KRAUSS, Irene, Ercole, Carmine, and Picone, Delia
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Multiple swapping ,RNase P ,Stereochemistry ,C-terminal 3D domain swapping ,Dimer ,Ribonuclease inhibitor ,Molecular Sequence Data ,Size-exclusion chromatography ,Biophysics ,Peptide ,Hinge peptide ,Crystallography, X-Ray ,Biochemistry ,chemistry.chemical_compound ,Structural Biology ,Endoribonucleases ,Hydrolase ,Genetics ,Animals ,Amino Acid Sequence ,Protein Structure, Quaternary ,Molecular Biology ,chemistry.chemical_classification ,Cell Biology ,Domain swapping ,Protein Structure, Tertiary ,chemistry ,Mutation ,Iodoacetamide ,Bovine seminal ribonuclease ,Cattle ,Protein quaternary structure ,Protein Multimerization ,Cytotoxic RNase - Abstract
Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111–116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.
- Published
- 2013
224. A novel interdomain interface in crystallins: structural characterization of the βγ-crystallin fromGeodia cydoniumat 0.99 Å resolution
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Filomena Sica, Marco Grassi, Alessandro Vergara, Antonello Merlino, Lelio Mazzarella, Elio Pizzo, Giuseppe D'Alessio, Vergara, Alessandro, M., Grassi, Sica, Filomena, Pizzo, Eliodoro, G., D'Alessio, L., Mazzarella, and Merlino, Antonello
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Models, Molecular ,Protein Folding ,Amino Acid Motifs ,Geodia cydonium ,Computational biology ,Crystallography, X-Ray ,Evolution, Molecular ,Structural Biology ,Crystallin ,Atomic resolution ,crystallin ,evolution ,Animals ,Geodia ,Binding site ,x-ray crystallography ,Binding Sites ,biology ,high resolution ,SUPERFAMILY ,General Medicine ,biology.organism_classification ,Crystallins ,eye diseases ,Sponge ,Crystallography ,Protein folding ,sense organs - Abstract
The [beta][gamma]-crystallin superfamily includes highly diverse proteins belonging to all of the kingdoms of life. Based on structural topology, these proteins are considered to be evolutionarily related to the long-lived [beta][gamma]-crystallins that constitute the vertebrate eye lens. This study reports the crystallographic structure at 0.99 Å resolution of the two-domain [beta][gamma]-crystallin (geodin) from the sponge Geodia cydonium. This is the most ancient member of the [beta][gamma]-crystallin superfamily in metazoans. The X-ray structure shows that the geodin domains adopt the typical [beta][gamma]-crystallin fold with a paired Greek-key motif, thus confirming the hypothesis that the crystallin-type scaffold used in the evolution of bacteria and moulds was recruited very early in metazoans. As a significant new structural feature, the sponge protein possesses a unique interdomain interface made up by pairing between the second motif of the first domain and the first motif of the second domain. The atomic resolution also allowed a detailed analysis of the calcium-binding site of the protein.
- Published
- 2013
225. The cold way for glutathione biosynthesis in the psychrophile Pseudoalteromonas haloplanktis. Redundancy and reaction rates
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Chiara Maranta, Filomena Sica, Alessandra Capasso, Rosario Rullo, Maria Rosaria Ruocco, Amalia De Angelis, Emmanuele De Vendittis, Antonella Albino, Albino, Antonella, DE ANGELIS, Amalia, Rullo, Rosario, Maranta, Chiara, Capasso, Alessandra, Ruocco, MARIA ROSARIA, Sica, Filomena, and DE VENDITTIS, Emmanuele
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0301 basic medicine ,chemistry.chemical_classification ,biology ,Stereochemistry ,General Chemical Engineering ,General Chemistry ,Glutathione ,biology.organism_classification ,Glutathione synthetase ,Pseudoalteromonas haloplanktis ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Enzyme ,Biochemistry ,Biosynthesis ,chemistry ,Cold adaptation, g-glutamyl-cysteine ligase, biochemical studies, spectroscopic measuraments ,ATP hydrolysis ,Thiol ,Cysteine - Abstract
This work describes major features and specificities of glutathione (GSH) biosynthesis in the cold-adapted Pseudoalteromonas haloplanktis. Two steps of ATP hydrolysis, catalysed by γ-glutamyl-cysteine ligase (PhGshA) and glutathione synthetase (PhGshB), drive GSH formation; however, differently from other sources, this psychrophile contains two redundant PhGshAs. The biochemical properties of recombinant rPhGshB and rPhGshA II were previously reported; here we report the characterization of rPhGshA I, together with a structural comparison with rPhGshA II. The availability of the three enzymes involved in GSH biosynthesis allowed the reconstitution of two systems producing this thiol in P. haloplanktis. Both systems were active, although with different reaction rates; in particular, rPhGshB worked more efficiently with rPhGshA I compared to rPhGshA II, also because of the different expression of the corresponding genes. However, the lower KM of rPhGshA II for cysteine compared to rPhGshA I could permit an effective GSH production even under conditions of low cysteine content. Coupling of the kinetics of GSH production with that of ATP hydrolysis was realised for the first time and proved the rigorous stoichiometry of GSH biosynthesis, resulting in two moles of ATP hydrolysed per one mole of GSH formed. Furthermore, after dissecting the reaction rates for GSH biosynthesis, we demonstrated that synthesis of this thiol was rate-limited by the step catalysed by rPhGshB, at least in the cold-adapted source. Therefore, the GshA rate-limited step postulated in other sources could be revised or restricted to conditions of limiting substrate amounts.
- Published
- 2016
226. Exploring the unfolding mechanism of γ-glutamyltranspeptidases: The case of the thermophilic enzyme from Geobacillus thermodenitrificans
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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
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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
227. Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding
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V. A. Spiridonova, Irene Russo Krauss, Filomena Sica, Andrea Pica, Valeria Napolitano, RUSSO KRAUSS, Irene, Vera, Spiridonova, Pica, Andrea, Napolitano, Valeria, and Sica, Filomena
- Subjects
0301 basic medicine ,Models, Molecular ,Aptamer ,Biology ,Bioinformatics ,G-quadruplex ,Crystallography, X-Ray ,Thrombin, apamer ,auplex/quadruplex, junction, anticoagulan sctivity, crystal structure ,03 medical and health sciences ,Thrombin ,Structural Biology ,Hydrolase ,Genetics ,medicine ,heterocyclic compounds ,Hydrogen bond ,Oligonucleotide ,Hydrogen Bonding ,Aptamers, Nucleotide ,G-Quadruplexes ,030104 developmental biology ,Duplex (building) ,Biophysics ,Nucleic Acid Conformation ,Corrigendum ,Anti-thrombin aptamers ,medicine.drug - Abstract
Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.
- Published
- 2015
228. Thrombin–aptamer recognition: a revealed ambiguity
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Lelio Mazzarella, Antonello Merlino, Concetta Giancola, Filomena Sica, Antonio Randazzo, Irene Russo Krauss, RUSSO KRAUSS, Irene, Merlino, Antonello, Giancola, Concetta, Randazzo, Antonio, Mazzarella, Lelio, and Sica, Filomena
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Oligonucleotide ,Aptamer ,Thrombin ,Rational design ,Aptamers, Nucleotide ,Biology ,Crystallography, X-Ray ,Combinatorial chemistry ,DNA-Binding Proteins ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biochemistry ,Structural Biology ,Genetics ,medicine ,Humans ,Molecule ,DNA ,Function (biology) ,medicine.drug - Abstract
Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human ?-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5'-5' polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin-mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin-aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.
- Published
- 2011
229. Biophysical and biochemical characterization of a liposarcoma-derived recombinant MnSOD protein acting as an anticancer agent
- Author
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Piero Pucci, Ettore Novellino, Antonella Schiattarella, Lelio Mazzarella, Filomena Sica, Paolo Grieco, Jean Rommelaere, R. Lorizio, Alessandro Sica, Franco Morelli, Roberto Mancini, Antonella Borrelli, Antonella Occhiello, Luigi Aloj, Michela Aurilio, Aldo Mancini, Daniela Pagnozzi, Alessandra Pica, A., Mancini, A., Borrelli, A., Schiattarella, L., Aloj, M., Aurilio, F., Morelli, Pica, Alessandra, Occhiello, Antonella, Lorizio, Roberto, R., Mancini, Sica, Alessandro, Mazzarella, Lelio, Sica, Filomena, Grieco, Paolo, Novellino, Ettore, D., Pagnozzi, Pucci, Pietro, and J., Rommelaere
- Subjects
Signal peptide ,Spectrometry, Mass, Electrospray Ionization ,Cancer Research ,antioxidant ,Biochemical Phenomena ,Molecular Sequence Data ,Biophysics ,Mice, Nude ,Antineoplastic Agents ,Biology ,Biochemistry ,Peptide Mapping ,Biophysical Phenomena ,Chromatography, Affinity ,rMnSOD ,law.invention ,Mice ,anticancer agent ,free radicals ,In vivo ,law ,Cell Line, Tumor ,Complementary DNA ,Gene expression ,Animals ,Humans ,Amino Acid Sequence ,Receptor ,free radical ,Base Sequence ,Estradiol ,Superoxide Dismutase ,Circular Dichroism ,Hydrogen Peroxide ,Liposarcoma ,Xenograft Model Antitumor Assays ,Recombinant Proteins ,In vitro ,Oncology ,Health ,Cell culture ,Recombinant DNA ,Sequence Analysis - Abstract
A recombinant MnSOD (rMnSOD) synthesized by specific cDNA clones derived from a liposarcoma cell line was shown to have the same sequence as the wild-type MnSOD expressed in the human myeloid leukaemia cell line U937, except for the presence of the leader peptide at the N-terminus. These results were fully confirmed by the molecular mass of rMnSOD as evaluated by ES/MS analysis (26662.7 Da) and the nucleotide sequence of the MnSOD cDNA. The role of the leader peptide in rMnSOD was investigated using a fluorescent and/or 68Gallium-labelled synthetic peptide. The labelled peptide permeated MCF-7 cells and uptake could be inhibited in the presence of an excess of oestrogen. In vivo it was taken up by the tumour, suggesting that the molecule can be used for both therapy and diagnosis. The in vitro and in vivo pharmacology tests confirmed that rMnSOD is only oncotoxic for tumour cells expressing oestrogen receptors. Pharmacokinetic studies in animals performed with 125I- and 131I-labelled proteins confirmed that, when administered systemically, rMnSOD selectively reached the tumour, where its presence was unambiguously demonstrated by scintigraphic and PET scans. PCR analysis revealed that Bax gene expression was increased and the Bcl2 gene was down regulated in MCF7 cells treated with rMnSOD, which suggests that the protein induces a pro-apoptotic mechanism. © 2008 Wiley-Liss, Inc.
- Published
- 2008
230. Crystallization and Preliminary X-Ray Diffraction Studies of a Psychrophilic Iron Superoxide Dismutase from Pseudoalteromonas haloplanktis
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Filomena Sica, Alessandro Vergara, Irene Russo Krauss, Emmanuele De Vendittis, Immacolata Castellano, Antonello Merlino, Merlino, Antonello, RUSSO KRAUSS, Irene, Castellano, I., De Vendittis, E., Vergara, Alessandro, and Sica, Filomena
- Subjects
biology ,Superoxide Dismutase ,Chemistry ,General Medicine ,Crystallography, X-Ray ,biology.organism_classification ,Biochemistry ,law.invention ,Pseudoalteromonas haloplanktis ,Superoxide dismutase ,Crystal ,Pseudoalteromonas ,Crystallography ,Structural Biology ,law ,X-ray crystallography ,biology.protein ,Molecular replacement ,Crystallization ,Dimerization ,Monoclinic crystal system - Abstract
The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P2(1) and diffract to 2.1 A resolution. Form I has unit-cell parameters a=45.49A b=103.63A c=50.37A beta=108.2 degrees and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a=50.48A b=103.78A c=90.25A beta=103.8 degrees and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperature.
- Published
- 2008
231. Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate
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Sante Capasso, Piero Pucci, Gennaro Marino, Diego Ingrosso, Filomena Sica, Patrizia Galletti, Caterina Manna, Galletti, Patrizia, Ingrosso, Diego, C., Manna, Sica, Filomena, S., Capasso, Pucci, Pietro, G., Marino, Galletti, P, Ingrosso, D, Manna, C, Capasso, S, and Marino, Gennaro
- Subjects
chemistry.chemical_classification ,Oligopeptide ,Stereochemistry ,Substrate (chemistry) ,Esters ,Peptide ,Tripeptide ,Fast atom bombardment ,Protein O-Methyltransferase ,Methylation ,Biochemistry ,Catalysis ,Mass Spectrometry ,Substrate Specificity ,Residue (chemistry) ,chemistry.chemical_compound ,chemistry ,Amide ,Protein Methyltransferases ,Binding site ,Oligopeptides ,Demethylation ,Methyl group - Abstract
Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595–602], while very little is know concerning the structural requirements of the protein substrate. In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11 503–11 511]; (2) the isoaspartyl residue is not recognized unless its α-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methylesterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.
- Published
- 2005
232. Crystal structure of the dimeric unswapped form of bovine seminal ribonuclease
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Filomena Sica, Renata Piccoli, C. De Lorenzo, A. Di Fiore, Adriana Zagari, Rita Berisio, Lelio Mazzarella, Berisio, R, Sica, Filomena, DE LORENZO, C, DI FIORE, A, Piccoli, R, Zagari, Adriana, Mazzarella, L., DE LORENZO, Claudia, Piccoli, Renata, and Mazzarella, Lelio
- Subjects
Male ,Models, Molecular ,Protein Conformation ,Ribonuclease inhibitor ,Biophysics ,Crystal structure ,Crystallography, X-Ray ,Bovine pancreatic ribonuclease ,Biochemistry ,Ribonuclease ,Ribonucleases ,Semen ,Structural Biology ,Hydrolase ,Genetics ,Animals ,Protein Isoforms ,Conformational dimorphism ,Protein Structure, Quaternary ,Molecular Biology ,chemistry.chemical_classification ,Three-dimensional domain swapping ,Molecular Structure ,biology ,Cell Biology ,Bovine seminal ribonuclease ,Protein Subunits ,Enzyme ,chemistry ,biology.protein ,Cattle ,Crystallization ,Dimerization - Abstract
Bovine seminal ribonuclease is a unique case of protein dimorphism, since it exists in two dimeric forms, with different biological and kinetic behavior, which interconvert into one another through three-dimensional swapping. Here we report the crystal structure, at 2.2 Å resolution, of the unswapped form of bovine seminal ribonuclease. Besides completing the structural definition of bovine seminal ribonuclease conformational dimorphism, this study provides the structural basis to explain the dependence of the enzyme cooperative effects on its swapping state.
- Published
- 2003
233. Structural study of a single-point mutant of Sulfolobus solfataricus alcohol dehydrogenase with enhanced activity
- Author
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Mosè Rossi, Ilaria Bruno, Carlo A. Raia, Luciana Esposito, Adriana Zagari, Antonietta Giordano, Filomena Sica, Lelio Mazzarella, Esposito, L, Bruno, I, Sica, Filomena, Raia, Ca, Giordano, A, Rossi, M, Mazzarella, L, and Zagari, A.
- Subjects
Models, Molecular ,Protein Conformation ,Enzyme activation ,ved/biology.organism_classification_rank.species ,Coenzymes ,Biophysics ,Biology ,Crystallography, X-Ray ,Biochemistry ,Cofactor ,Sulfolobus ,Enzyme activator ,Structural Biology ,Oxidoreductase ,Single mutation ,Coenzyme binding ,Genetics ,Point Mutation ,Molecular Biology ,Alcohol dehydrogenase ,chemistry.chemical_classification ,ved/biology ,Crystal structure ,Sulfolobus solfataricus ,Cell Biology ,Enzyme ,chemistry ,biology.protein ,NAD+ kinase - Abstract
Alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) is the only enzyme from Archaea among the structurally studied members of the medium-chain ADH family described so far. Here, we present the three-dimensional structure of the apo form of the mutant N249Y which exhibits increased catalytic activity when compared to the wild-type enzyme. The substitution, located in the coenzyme binding domain, decreases the affinity for NAD(H) cofactor. The rearrangement of segments 248–250 and 270–275, induced by the mutation, suggests an explanation for the lower coenzyme affinity. This study also highlights the role in SsADH catalysis of the flexible loops located at the interface between the catalytic and the coenzyme domains.
- Published
- 2003
234. Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability
- Author
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Antonello Merlino, Maria Rosaria Ruocco, Irene Russo Krauss, Immacolata Castellano, Emmanuele De Vendittis, Bianca Rossi, Filomena Sica, Alessandra Capasso, Merlino, Antonello, RUSSO KRAUSS, Irene, Castellano, I, Ruocco, MARIA ROSARIA, Capasso, Alessandra, DE VENDITTIS, Emmanuele, Rossi, B, and Sica, Filomena
- Subjects
Models, Molecular ,Protein Denaturation ,Circular dichroism ,crystal structure ,Protein Conformation ,Static Electricity ,Mutant ,Biophysics ,Superoxide dismutase ,Unfolding ,Crystallography, X-Ray ,Biochemistry ,Analytical Chemistry ,Pseudoalteromonas haloplanktis ,03 medical and health sciences ,Protein structure ,Native state ,Denaturation (biochemistry) ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,Protein Stability ,Chemistry ,Circular Dichroism ,030302 biochemistry & molecular biology ,Wild type ,Psychrophilic enzyme ,Thermal stability ,biology.organism_classification ,Cold Temperature ,Spectrometry, Fluorescence ,Mutation ,biology.protein - Abstract
A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge-charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme.
- Published
- 2014
235. The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with GDP reveals novel features in nucleotide binding and exchange
- Author
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Mariorosario Masullo, Luigi Vitagliano, Vincenzo Bocchini, Adriana Zagari, Filomena Sica, Vitagliano, L., Masullo, M., Sica, Filomena, Zagari, Adriana, Bocchini, V., and Zagari, A.
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archaeal EF-1 ,crystal structure ,magnesium ion ,Stereochemistry ,Archaeal Proteins ,ved/biology.organism_classification_rank.species ,Biology ,Guanosine Diphosphate ,Article ,General Biochemistry, Genetics and Molecular Biology ,Sulfolobus ,Nucleotide exchange factor ,Peptide Elongation Factor 1 ,Protein structure ,Magnesium ,Archaeal EF-1, Crystal structure, magnesium ion, nucleotide exchange, Sulfolobus solfataricus ,Binding site ,Molecular Biology ,Magnesium ion ,nucleotide exchange ,Binding Sites ,Crystallography ,General Immunology and Microbiology ,ved/biology ,General Neuroscience ,Sulfolobus solfataricus ,GDP binding ,Guanine Nucleotides ,Protein Structure, Tertiary ,Elongation factor ,Biochemistry ,EF-Tu - Abstract
The crystal structure of elongation factor 1alpha from the archaeon Sulfolobus solfataricus in complex with GDP (SsEF-1alpha.GDP) at 1.8 A resolution is reported. As already known for the eubacterial elongation factor Tu, the SsEF-1alpha.GDP structure consists of three different structural domains. Surprisingly, the analysis of the GDP-binding site reveals that the nucleotide- protein interactions are not mediated by Mg(2+). Furthermore, the residues that usually co-ordinate Mg(2+) through water molecules in the GTP-binding proteins, though conserved in SsEF-1alpha, are located quite far from the binding site. [(3)H]GDP binding experiments confirm that Mg(2+) has only a marginal effect on the nucleotide exchange reaction of SsEF-1alpha, although essential to GTPase activity elicited by SsEF-1alpha. Finally, structural comparisons of SsEF- 1alpha.GDP with yeast EF-1alpha in complex with the nucleotide exchange factor EF-1beta shows that a dramatic rearrangement of the overall structure of EF-1alpha occurs during the nucleotide exchange.
- Published
- 2001
236. Crystallization of multiple forms of bovine seminal ribonuclease in the liganded and unliganded state
- Author
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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
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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
237. Duplex-quadruplex motifs in a peculiar structural organization cooperatively contribute to thrombin binding of a DNA aptamer
- Author
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Irene Russo Krauss, Lelio Mazzarella, Filomena Sica, Andrea Pica, Antonello Merlino, RUSSO KRAUSS, Irene, Pica, Andrea, Merlino, Antonello, L., Mazzarella, and Sica, Filomena
- Subjects
Models, Molecular ,Aptamer ,TBA analogue ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,quadruple eliche di DNA ,Thrombin ,Structural Biology ,medicine ,Humans ,heterocyclic compounds ,Base sequence ,A-DNA ,Structural organization ,Base Sequence ,Oligonucleotide ,General Medicine ,Aptamers, Nucleotide ,Biochemistry ,Duplex (building) ,Biophysics ,Nucleic Acid Conformation ,circulatory and respiratory physiology ,medicine.drug ,Protein Binding - Abstract
Potent second-generation thrombin aptamers adopt a duplex–quadruplex bimodular folding and recognize thrombin exosite II with very high affinity and specificity. A sound model of these oligonucleotides, either free or in complex with thrombin, is not yet available. Here, a structural study of one of these aptamers, HD22-27mer, is presented. The crystal structure of this aptamer in complex with thrombin displays a novel architecture in which the helical stem is enchained to a pseudo-G-quadruplex. The results also underline the role of the residues that join the duplex and quadruplex motifs and control their recruitment in thrombin binding.
- Published
- 2013
238. Probing the activity of diguanylate cyclases and c-di-GMP phosphodiesterases in real-time by CD spectroscopy
- Author
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Filomena Sica, Silvia Fernicola, Stefano Franceschini, Giorgio Giardina, Andrea Pica, Annegret Brandt, Valentina Stelitano, Serena Rinaldo, Francesca Cutruzzolà, Department of Biochemical Sciences 'Rossi Fanelli', Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Department of Chemical Sciences, Università degli studi di Napoli Federico II, Institute of Biostructure and Bioimaging, CNR - Naples, Ministero della Universita e Ricerca of Italy [RBRN07BMCT and 20094BJ9R7 to F.C, RBFR10LHD1 to S.R.], Sapienza University of Rome, Fondazione Italiana Fibrosi Cistica [13/2009- with the contribution of Delegazione Novara and Delegazione Cosenza2 to F.C.]. Funding for open access charge: Ministero della Universita e Ricerca of Italy, Stelitano, V, Brandt, A, Fernicola, S, Franceschini, S, Giardina, G, Pica, Andrea, Rinaldo, S, Sica, Filomena, Cutruzzolà, F., Department of Biochemical Sciences, Università degli Studi di Roma 'La Sapienza' [Rome] - Réseau International des Instituts Pasteur - Institut Pasteur - Fondation Cenci Bolognetti, and Università degli studi di Napoli Federico II [Napoli]
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Models, Molecular ,Circular dichroism ,Dimer ,MESH: Guanylate Cyclase ,MESH: Manganese ,Biology ,010402 general chemistry ,01 natural sciences ,MESH: Circular Dichroism ,03 medical and health sciences ,chemistry.chemical_compound ,3',5'-Cyclic-GMP Phosphodiesterases ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Genetics ,MESH: Cyclic GMP ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Cyclic GMP ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Manganese ,Circular Dichroism ,Biofilm ,Phosphodiesterase ,biology.organism_classification ,MESH: 3',5'-Cyclic-GMP Phosphodiesterases ,In vitro ,0104 chemical sciences ,Enzyme ,chemistry ,Biochemistry ,Guanylate Cyclase ,Methods Online ,Bacteria ,MESH: Models, Molecular ,Dinucleoside Phosphates - Abstract
International audience; Bacteria react to adverse environmental stimuli by clustering into organized communities called biofilms. A remarkably sophisticated control system based on the dinucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) is involved in deciding whether to form or abandon biofilms. The ability of c-di-GMP to form self-intercalated dimers is also thought to play a role in this complex regulation. A great advantage in the quest of elucidating the catalytic properties of the enzymes involved in c-di-GMP turnover (diguanylate cyclases and phosphodiesterases) would come from the availability of an experimental approach for in vitro quantification of c-di-GMP in real-time. Here, we show that c-di-GMP can be detected and quantified by circular dichroism (CD) spectroscopy in the low micromolar range. The method is based on the selective ability of manganese ions to induce formation of the intercalated dimer of the c-di-GMP dinucleotide in solution, which displays an intense sigmoidal CD spectrum in the near-ultraviolet region. This characteristic spectrum originates from the stacking interaction of the four mutually intercalated guanines, as it is absent in the other cyclic dinucleotide 3'-5' cyclic adenilic acid (c-di-AMP). Thus, near-ultraviolet CD can be used to effectively quantify in real-time the activity of diguanylate cyclases and phosphodiesterases in solution.
- Published
- 2013
239. An overview of biological macromolecule crystallization
- Author
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Irene Russo Krauss, Antonello Merlino, Filomena Sica, Alessandro Vergara, RUSSO KRAUSS, Irene, Merlino, Antonello, Vergara, Alessandro, and Sica, Filomena
- Subjects
crystallization ,Macromolecular Substances ,Nanotechnology ,Review ,new methodologies ,Catalysis ,law.invention ,Inorganic Chemistry ,lcsh:Chemistry ,X-Ray Diffraction ,Atomic resolution ,law ,Nucleic Acids ,protein, nucleic acid, membrane, crystallization, new methodologies ,Physical and Theoretical Chemistry ,Crystallization ,Molecular Biology ,lcsh:QH301-705.5 ,membrane ,Spectroscopy ,Neutrons ,Chemistry ,Organic Chemistry ,Biological macromolecule ,Membrane Proteins ,General Medicine ,Computer Science Applications ,nucleic acid ,lcsh:Biology (General) ,lcsh:QD1-999 ,Protein crystallization ,protein - Abstract
The elucidation of the three dimensional structure of biological macromolecules has provided an important contribution to our current understanding of many basic mechanisms involved in life processes. This enormous impact largely results from the ability of X-ray crystallography to provide accurate structural details at atomic resolution that are a prerequisite for a deeper insight on the way in which bio-macromolecules interact with each other to build up supramolecular nano-machines capable of performing specialized biological functions. With the advent of high-energy synchrotron sources and the development of sophisticated software to solve X-ray and neutron crystal structures of large molecules, the crystallization step has become even more the bottleneck of a successful structure determination. This review introduces the general aspects of protein crystallization, summarizes conventional and innovative crystallization methods and focuses on the new strategies utilized to improve the success rate of experiments and increase crystal diffraction quality.
- Published
- 2013
240. Dissecting the contribution of thrombin exosite I in the recognition of thrombin binding aptamer
- Author
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Filomena Sica, Naoki Sugimoto, Irene Russo Krauss, Satoru Nagatoishi, Antonello Merlino, Andrea Pica, Pica, Andrea, RUSSO KRAUSS, Irene, Merlino, Antonello, S., Nagatoishi, N., Sugimoto, and Sica, Filomena
- Subjects
Oligonucleotide ,Stereochemistry ,Circular Dichroism ,Aptamer ,Thrombin ,Rational design ,Cell Biology ,Aptamers, Nucleotide ,Antiparallel (biochemistry) ,G-quadruplex ,Biochemistry ,TBA ,Nucleobase ,G-Quadruplexes ,chemistry.chemical_compound ,TBA analogues ,chemistry ,Mutation ,medicine ,Humans ,Crystallization ,Molecular Biology ,DNA ,medicine.drug - Abstract
Thrombin plays a pivotal role in the coagulation cascade; therefore, it represents a primary target in the treatment of several blood diseases. The 15-mer DNA oligonucleotide, 5'-GGTTGGTGTGGTTGG-3', known as Thrombin Binding Aptamer (TBA), is a highly potent inhibitor of the enzyme. TBA folds as an antiparallel chair-like G-quadruplex structure, with two G-tetrads surrounded by two TT loops on one side and a TGT loop on the opposite side. Previous crystallographic studies have shown that TBA binds thrombin exosite I by its TT loops, T3T4 and T12T13. In order to get a better understanding of the thrombin-TBA interaction, we have undertaken a crystallographic characterization of the complexes between thrombin and two TBA mutants, TBA??T3 and TBA??T12, which lack the nucleobase of T3 and T12, respectively. The structural details of the two complexes show that exosite I is actually split into two regions, which contribute differently to TBA recognition. These results provide the basis for a more rational design of new aptamers with improved therapeutic action. This article is protected by copyright. All rights reserved.
- Published
- 2013
241. Deamidation in Proteins: The Crystal Structure of Bovine Pancreatic Ribonuclease with an Isoaspartyl Residue at Position 67
- Author
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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
242. Effect of NaCl on the conformational stability of the thermophilic γ-glutamyltranspeptidase from Geobacillus thermodenitrificans: Implication for globular protein halotolerance
- Author
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Giuseppe Graziano, Francesco La Cara, Antonello Merlino, Andrea Pica, Immacolata Castellano, Irene Russo Krauss, Filomena Sica, Pica, Andrea, RUSSO KRAUSS, Irene, Castellano, I., La Cara, F., Graziano, G, Sica, Filomena, and Merlino, Antonello
- Subjects
chemistry.chemical_classification ,Circular dichroism ,Quenching (fluorescence) ,Chemistry ,Globular protein ,Biophysics ,Geobacillus ,gamma-Glutamyltransferase ,Sodium Chloride ,Biochemistry ,Protein Structure, Secondary ,Analytical Chemistry ,Protein structure ,Bacterial Proteins ,Enzyme Stability ,Halotolerance ,Denaturation (biochemistry) ,Protein stabilization ,Molecular Biology ,Protein secondary structure - Abstract
The transpeptidation activity of γ-glutamyltranspeptidase from Geobacillus thermodenitrificans (GthGT) is 27 negligible and the enzyme is highly thermostable. Here we have examined the effect of concentrated NaCl 28 solutions on structure, stability, dynamics and enzymatic activity of GthGT. The protein exhibited hydrolytic 29 activity over a broad range of NaCl concentrations. Even at 4.0 M NaCl, GthGT retained more than 90% of the 30 initial activity and showed unaltered fluorescence emission, secondary structure and acrylamide quenching 31 on tryptophan fluorescence. Furthermore, at 2.8 M and 4.0 M NaCl the temperature-induced unfolding pro- 32 files are dramatically changed with large (>20 °C) positive shifts in the denaturation temperature. These fea- 33 tures make GthGT an ideal system to be used in industrial processes that require high temperatures and 34 high-salt environments. A general explanation of the NaCl effect by means of a statistical thermodynamic 35 model is also provided, together with an analysis of residue distribution between protein surface and interior 36 in 15 non-redundant families of halophilic and non-halophilic proteins. The results are in line with a compar- 37 ative sequence and structural analysis between halophilic and non-halophilic γ-glutamyltranspeptidases 38 which revealed that a major role in halotolerance should be played by solvent exposed negatively charged 39 residues.
- Published
- 2012
243. High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity
- Author
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Antonio Randazzo, Lelio Mazzarella, Filomena Sica, Irene Russo Krauss, Ettore Novellino, Antonello Merlino, RUSSO KRAUSS, Irene, Merlino, Antonello, Randazzo, Antonio, Novellino, Ettore, Mazzarella, Lelio, and Sica, Filomena
- Subjects
Models, Molecular ,Circular dichroism ,genetic structures ,Aptamer ,thrombing binding aptamer ,Ionic bonding ,High resolution ,Biology ,Inhibitory postsynaptic potential ,Crystallography, X-Ray ,Ion ,X-ray ,Thrombin ,Structural Biology ,Cations ,Genetics ,medicine ,Thrombin-binding aptamer ,Circular Dichroism ,Sodium ,Aptamers, Nucleotide ,Biochemistry ,Biophysics ,Potassium ,medicine.drug - Abstract
The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human ??-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K(+) ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of ??-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin-TBA complex formed in the presence of Na(+) or K(+) and a circular dichroism study of the structural stability of the aptamer both free and complexed with ??-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na(+) and K(+) on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein-aptamer interface. The present data, in combination with those previously obtained on the complex between ??-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement.
- Published
- 2012
244. Destabilization of Lipid Membranes by a Peptide Derived from Glycoprotein gp36 of Feline Immunodeficiency Virus: A Combined Molecular Dynamics/Experimental Study
- Author
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Gerardino D'Errico, Luigi Paduano, Filomena Sica, Elena Busi, Giovanna Fragneto, Manuela Grimaldi, Giuseppe Vitiello, Anna Maria D'Ursi, Riccardo Basosi, Antonello Merlino, Merlino, Antonello, Vitiello, Giuseppe, M., Grimaldi, Sica, Filomena, E., Busi, R., Basosi, A., D’Ursi, G., Fragneto, Paduano, Luigi, and D'Errico, Gerardino
- Subjects
TRANSMEMBRANE GLYCOPROTEIN ,PROXIMAL REGION ,Lipid Bilayers ,STRUCTURAL-ANALYSIS ,Model lipid bilayer ,Immunodeficiency Virus, Feline ,Molecular Dynamics Simulation ,proteins/peptides and phospholipid bilayers interactions ,COMPUTER-SIMULATION ,MAGNETIC-RESONANCE ,Materials Chemistry ,Membrane fluidity ,Lipid bilayer phase behavior ,Physical and Theoretical Chemistry ,Lipid bilayer ,Glycoproteins ,DYNAMICS SIMULATIONS ,PHOSPHOLIPID-BILAYERS ,NEUTRON REFLECTIVITY ,ROMATIC RESIDUES ,RIBONUCLEASE-A ,Chemistry ,Bilayer ,molecular dynamic ,Circular Dichroism ,Peripheral membrane protein ,Electron Spin Resonance Spectroscopy ,Lipid bilayer fusion ,Biological membrane ,Hydrogen Bonding ,Surfaces, Coatings and Films ,viral fusion protein ,Crystallography ,Spectrometry, Fluorescence ,Phosphatidylcholines ,EPR ,Hydrophobic and Hydrophilic Interactions ,Oligopeptides - Abstract
Viral fusion glycoproteins present a membrane-proximal external region (MPER) which is usually rich in aromatic residues and exhibits a marked tendency to stably reside at the membrane interfaces, leading, through unknown mechanisms, to a destabilization of the bilayer structure. This step has been proposed to be fundamental for the fusion process between target membrane and viral envelope. In the present work, we investigate the interaction between an octapeptide (C8) deriving from the MPER domain of gp36 of feline immunodeficiency virus and POPC bilayers by combining experimental results obtained by neutron reflectivity, electron spin resonance, circular dichroism, and fluorescence spectroscopy with molecular dynamics simulations. Our data indicate that C8 binds to the lipid bilayer adsorbing onto the membrane surface without deep penetration. As a consequence of this interaction, the bilayer thickness decreases. The association of the peptide with the lipid membrane is driven by hydrogen bonds as well as hydrophobic interactions that the Trp side chains form with the lipid headgroups. Upon peptide-bilayer interaction, C8 forms transient secondary structures ranging from 3(10) helices to turn conformations, while acyl chains of the peptide-exposed POPC molecules assume a more ordered packing. At the same time, lipid headgroups' hydration increases. The asymmetric lipid bilayer perturbation is proposed to play a fundamental role in favoring the membrane fusion process.
- Published
- 2012
245. Increasing the X-ray Diffraction Power of Protein Crystals by Dehydration: The Case of Bovine Serum Albumin and a Survey of Literature Data
- Author
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Carlo Andrea Mattia, Irene Russo Krauss, Antonello Merlino, Filomena Sica, RUSSO KRAUSS, Irene, Sica, Filomena, Mattia, Ca, and Merlino, Antonello
- Subjects
protein crystallization ,serum albumin ,Serum albumin ,crystal dehydration ,Crystal structure ,Crystallography, X-Ray ,Catalysis ,law.invention ,lcsh:Chemistry ,Inorganic Chemistry ,X-Ray Diffraction ,law ,medicine ,Animals ,Dehydration ,Amino Acid Sequence ,Physical and Theoretical Chemistry ,Crystallization ,Bovine serum albumin ,lcsh:QH301-705.5 ,Molecular Biology ,Spectroscopy ,X-ray crystallography ,biology ,Chemistry ,Communication ,Organic Chemistry ,Serum Albumin, Bovine ,General Medicine ,medicine.disease ,Human serum albumin ,Computer Science Applications ,Crystallography ,lcsh:Biology (General) ,lcsh:QD1-999 ,biology.protein ,Cattle ,Protein crystallization ,post-crystallization treatment ,medicine.drug ,crystal quality - Abstract
Serum albumin is one of the most widely studied proteins. It is the most abundant protein in plasma with a typical concentration of 5 g/100 mL and the principal transporter of fatty acids in plasma. While the crystal structures of human serum albumin (HSA) free and in complex with fatty acids, hemin, and local anesthetics have been characterized, no crystallographic models are available on bovine serum albumin (BSA), presumably because of the poor diffraction power of existing hexagonal BSA crystals. Here, the crystallization and diffraction data of a new BSA crystal form, obtained by the hanging drop method using MPEG 5K as precipitating agent, are presented. The crystals belong to space group C2, with unit-cell parameters a = 216.45 Å, b = 44.72 Å, c = 140.18 Å, β = 114.5°. Dehydration was found to increase the diffraction limit of BSA crystals from ~8 Å to 3.2 Å, probably by improving the packing of protein molecules in the crystal lattice. These results, together with a survey of more than 60 successful cases of protein crystal dehydration, confirm that it can be a useful procedure to be used in initial screening as a method of improving the diffraction limits of existing crystals.
- Published
- 2012
246. Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease
- Author
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Filomena Sica, Delia Picone, Carmine Ercole, Anna Balsamo, Antonello Merlino, Merlino, Antonello, Picone, Delia, Ercole, Carmine, Balsamo, A, and Sica, Filomena
- Subjects
Protein structure, 3D domain swapping, Cis-Pro, Structural stability, Protein mutations ,Protein Folding ,Magnetic Resonance Spectroscopy ,RNase P ,Dimer ,Peptide ,Bovine pancreatic ribonuclease ,Biochemistry ,RNase PH ,Protein Structure, Secondary ,chemistry.chemical_compound ,Protein structure ,X-Ray Diffraction ,Hydrolase ,Animals ,chemistry.chemical_classification ,biology ,Protein Stability ,Ribonuclease, Pancreatic ,General Medicine ,Protein Structure, Tertiary ,Crystallography ,Monomer ,chemistry ,Mutation ,biology.protein ,Cattle ,Dimerization - Abstract
3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1–13) and/or the C-terminal β-strand (residues 116–124), thus forming a variety of oligomers, including two different dimers. Cis - trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis - trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.
- Published
- 2012
247. Identification of an active dimeric intermediate populated during the unfolding process of the cambialistic superoxide dismutase from Streptococcus mutans
- Author
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Antonello Merlino, Alberto De Vendittis, Salvatore Marco, Irene Russo Krauss, Filomena Sica, Alessandro Vergara, Emmanuele De Vendittis, Bianca Rossi, Merlino, Antonello, RUSSO KRAUSS, Irene, Rossi, B., Vergara, Alessandro, De Vendittis, A., Marco, Salvatore, DE VENDITTIS, Emmanuele, and Sica, Filomena
- Subjects
Circular dichroism ,Protein Folding ,Protein Data Bank (RCSB PDB) ,Unfolding ,Biochemistry ,Superoxide dismutase ,Streptococcus mutans ,chemistry.chemical_compound ,Bacterial Proteins ,Guanidine ,Thermostability ,chemistry.chemical_classification ,biology ,Dimeric intermediate ,Superoxide Dismutase ,Circular Dichroism ,General Medicine ,Thermal stability ,biology.organism_classification ,Enzyme ,chemistry ,biology.protein ,Chemical stability - Abstract
Superoxide dismutases are enzymes that protect biological systems against oxidative damage caused by superoxide radicals. In this paper, a detailed characterization is presented on the stability of SmSOD, the dimeric cambialistic superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans, towards temperature and guanidine hydrochloride. Thermal and chemical denaturations were investigated by means of circular dichroism, fourth-derivative UV spectroscopy and fluorescence measurements. Data indicate that SmSOD is endowed with a significant thermostability and that both its thermal and guanidine hydrochloride-induced unfolding processes occur through a three-state model, characterized by a catalytically active dimeric intermediate species. To our knowledge, SmSOD is the smallest known dimeric protein that populates a well-structured active dimeric rather than a monomeric intermediate during unfolding processes.
- Published
- 2011
248. Monitoring Preparation of Derivative Protein Crystals via Raman Microscopy
- Author
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Filomena Sica, Antonello Merlino, Alessandro Vergara, Annamalai CHANDRASEKARAN, Merlino, Antonello, Sica, Filomena, and Vergara, Alessandro
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Supersaturation ,Cristallografia ,Materials science ,Drop (liquid) ,Nucleation ,law.invention ,Solvent ,Crystallography ,law ,Molecule ,Crystallization ,Protein crystallization ,proteine ,Raman ,Macromolecule - Abstract
Below to crystallographic applications, protein crystals are of great interest in other numerous fields of biology and biotechnology. Cataract, or the loss of transparency of the eye lens, is related to the alteration of physical, chemical, and structural properties of proteins of the crystallin family, that may lead to crystallization under some physiological conditions (Tardieu, 1998). Other pathological states are known to be a consequence of the in vivo formation of crystals, made of either proteins or other macromolecular assemblies. Examples are viral proteins stored in plant cells, viral particles in animal cells, hemoglobin C and S causing anemia, or ribosomal particles accumulating in the brain of patients suffering from presenile dementia (McPherson, 1999). Of course, crystals of biological macromolecules that are prepared in vitro have important applications: they are tools to obtain atomic models of the molecules and to design specific ligands and new drug formulations. Medicinal formulations composed of either insulin (Richards et al., 1999) or α-interferon crystals (Reichert et al., 1996) are already applied in treatments to ensure the continuous release of protein in blood. Crystallographic analysis of highly ordered crystals with intense X-ray sources provides accurate three dimensional structures (Ducruix & Giege, 1999). The success of this technique strictly depends on obtaining diffraction-quality crystals. The process of crystallization remains a hit-and-miss affair, typically involving screening hundreds of conditions. The crystallization of biological macromolecules shares many common properties with those of small solute molecules (e.g. growth by 2D nucleation or by screw dislocation mechanisms), but their crystals exhibit several peculiarities: most of them have a high solvent content (e.g. 30-80 vol%), few intermolecular contacts, and a high density of defects (Malkin et al., 1996). Briefly, protein crystallization requires the formation of a supersaturated protein-precipitant solution. The transition from a stable solution to a supersaturated one can be achieved by increasing the concentration of precipitant and/or that of protein (Vergara et al., 2003). The most frequently used crystallization method is the vapor diffusion technique. A drop containing protein, buffer, salt and precipitant is equilibrated against a reservoir (buffer, salt and precipitant). The difference in concentration between the drop (lower) and the reservoir (higher) drives the system toward equilibrium by diffusion through the vapor phase. The drop can either be placed on the underside of the cover slide (hanging drop) or placed on a
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- 2011
249. Protonation of histidine 55 affects the oxygen access to heme in the alpha chain of the hemoglobin from the Antarctic fish Trematomus bernacchii
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Filomena Sica, Marcelo A. Martí, Leonardo Boechi, Antonello Merlino, Alessandro Vergara, Darío A. Estrin, Lelio Mazzarella, Boechi, L., Martì, M. A., Vergara, Alessandro, Sica, Filomena, Mazzarella, Lelio, Estrin, D. A., and Merlino, Antonello
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Models, Molecular ,Stereochemistry ,Clinical Biochemistry ,Antarctic Regions ,Cooperativity ,Heme ,Biochemistry ,chemistry.chemical_compound ,Hemoglobins ,Trematomus ,Genetics ,Animals ,Histidine ,Molecular Biology ,biology ,Root effect ,Cell Biology ,biology.organism_classification ,Protein tertiary structure ,Oxygen ,chemistry ,Trematomus bernacchii, * hemoglobin, * implicit ligand sampling, * molecular dynamics simulations, * pH, * oxygen binding ,Hemoglobin ,Protons ,Oxygen binding - Abstract
The Root effect describes the drastic drop of oxygen affinity and loss of cooperativity at acidic pH expressed in the hemoglobins (Hb) of certain fish. The comparison between the deoxy structures of the Root effect Hb from the Antarctic fish Trematomus bernacchii (HbTb) at different pHs (pH = 6.2 and pH = 8.4) shows that the most significant differences are localized at the CDα region, where a salt bridge between Asp48 and His55 breaks during the low-to-high pH transition. In order to shed light on the relationship between pH, CDα loop structure and dynamics, and oxygen access to the active site in the alpha chain of HbTb, different computer simulation techniques were performed. Our results highlight the importance of the protonation of His55 in regulating oxygen access, underscoring its pivotal role in the structural and functional properties of HbTb. These data provide further support to the hypothesis that this residue might contribute to the release of Root protons in HbTb and underline the fact that an efficient transport of molecular oxygen in Hbs relies on a subtle balance of tertiary structure and protein conformational flexibility. © 2011 IUBMB IUBMB Life, 63(3): 175–182, 2011
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- 2011
250. IMPROVING PROTEIN CRYSTAL QUALITY BY THE WITHOUT-OIL MICROBATCH METHOD: CRYSTALLIZATION AND PRELIMINARY X-RAY DIFFRACTION ANALYSIS OF GLUTATHIONE SYNTHETASE FROM PSEUDOALTEROMONAS HALOPLANKTIS
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Irene Russo Krauss, Emmanuele De Vendittis, Andrea Pica, Mariorosario Masullo, Alessandro Vergara, Antonella Albino, Filomena Sica, Antonello Merlino, Merlino, Antonello, RUSSO KRAUSS, Irene, Albino, Antonella, Pica, Andrea, Vergara, Alessandro, Masullo, Mariorosario, DE VENDITTIS, Emmanuele, and Sica, Filomena
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glutathione synthetase ,psychrophile ,without-oil microbatch ,Glutathione Synthase ,Catalysis ,law.invention ,Pseudoalteromonas haloplanktis ,Inorganic Chemistry ,Bacterial protein ,lcsh:Chemistry ,Pseudoalteromonas ,Bacterial Proteins ,X-Ray Diffraction ,law ,Physical and Theoretical Chemistry ,Crystallization ,Molecular Biology ,lcsh:QH301-705.5 ,Spectroscopy ,X-ray crystallography ,biology ,crystal quality ,Chemistry ,Communication ,Organic Chemistry ,General Medicine ,biology.organism_classification ,Recombinant Proteins ,Glutathione synthetase ,Computer Science Applications ,Crystallography ,lcsh:Biology (General) ,lcsh:QD1-999 ,Electrophoresis, Polyacrylamide Gel ,Protein crystallization - Abstract
Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from l-γ-glutamyl- l-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhGshB) has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 Å resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 83.28 Å, b = 119.88 Å, c = 159.82 Å. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date.
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- 2011
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