1,721,038 research outputs found
The three-dimensional crystal structure of carp fish egg lectin
No full textUna nuova lectina è stata recentemente scoperta ed isolata nel nostro
laboratorio dalle uova di carpa ed è stata denominata Fish Egg Lectin (FEL). FEL
è una glicoproteina di 238 aminoacidi dal peso molecolare di 26.6 kD e l’analisi
della sequenza ha mostrato una certa omologia con una lectina isolata dal plasma
del granchio Tachypleus tridentatus, la Tachylectin 1, che è coinvolta nella
risposta immunitaria aspecifica di questo invertebrato. Studi biochimici eseguiti dal
gruppo della Prof. Galliano (Università di Pavia) hanno evidenziato che questa
lectina è monomerica in soluzione e che è in grado di legare i carboidrati presenti
sulla parete di cellule batteriche sia Gram positive che negative. Tra i
monosaccaridi, la più alta affinità è stata riscontrata verso la N-acetilglucosamina
e la N-acetilgalattosamina mentre sembra essere molto bassa o nulla nei confronti
dei corrispondenti carboidrati non acetilati e verso i disaccaridi come il lattosio.
Saggi immunoistochimici hanno dimostrato inoltre che la FEL è una proteina
specifica delle uova ed è assente in tutti gli altri tessuti. Nonostante ciò è la prima
volta che una lectina di questo tipo viene descritta in un animale vertebrato (può
essere considerata come la prima di una nuova famiglia) e il suo reale ruolo
fisiologico risulta ancora da chiarire.
l lavoro di questa tesi si è focalizzato sullo studio strutturale della Fish Egg
Lectin di carpa mediante diffrazione di raggi X. Numerose prove di cristallizzazione
sono state preparate con la proteina nativa fino ad ottenere cristalli singoli adatti
agli studi di diffrazione. I cristalli migliori sono stati ottenuti con la tecnica della
diffusione di vapore utilizzando una miscela di 15% 2-propanolo, 20% PEG 6000,
0.1 M HEPES pH 7.5 come precipitante. Dall’analisi dei dati di diffrazione si è
potuto stabilire che tali cristalli sono ortorombici e che appartengono al gruppo
spaziale P212121, con parametri di cella a = 44.72 Å, b = 72.28 Å e c = 167.59 Å.
Non essendo possibile risolvere il problema della fase per sostituzione
molecolare (non è stata risolta finora nessuna struttura di proteine ritenute
omologhe alla FEL), si è dovuto procedere con il metodo classico della
sostituzione isomorfa multipla. Numerosi derivati sono stati preparati facendo
reagire i cristalli della proteina con vari composti di atomi pesanti ed i dati di
diffrazione sono stati raccolti ed analizzati. Fra questi, i migliori tre (quelli ottenuti
con K2PtCl4, Hg(Ac)2 e UO2(Ac)2) sono stati utilizzati per il calcolo delle fasi dei
fattori di struttura. Sono quindi state calcolate le mappe di densità elettronica a 2.5
Å di risoluzione ed il modello molecolare è stato costruito e raffinato.
Nell’unità asimmetrica questa lectina si presenta come un omodimero in cui
ciascun monomero è un dominio “six β-propeller”, una struttura simmetrica
costituita in prevalenza da β-strands antiparalleli che nel suo insieme ricorda
un’elica con sei pale con un canale centrale. L’analisi dei contatti intermolecolari
ed i calcoli sulla superficie di contatto fra i monomeri inducono a supporre che la
formazione del dimero non sia dovuta semplicemente all’impacchettamento nel
cristallo ma che questa abbia invece un significato funzionale (in contrasto con i
risultati pubblicati in precedenza). Un sito di legame per lo ione calcio è presente
nella cavità centrale di ogni monomero .
Allo scopo di identificare i siti di legame per i carboidrati, alcuni cristalli della
proteina nativa sono stati immersi in una soluzione del liquido cristallizzante
contenente 0.1 M N-acetilglucosamina ed i dati di diffrazione raccolti ed analizzati.
Un singolo sito di legame è stato identificato con certezza su uno solo dei due
monomeri, in contrasto con la presenza di siti multipli descritta in altre due lectine
con struttura a β-propeller. L’evidenza di un unico sito (per di più in un omodimero)
è con maggior probabilità dovuta al metodo utilizzato per la preparazione dei
cristalli del complesso o alla bassa affinità per il ligando utilizzato piuttosto che ad
una reale peculiarità di questa proteina. La modalità di legame della N-
acetilglucosamina ricorda quella descritta per un’altra lectina di Tachypleus
tridentatus, Tachylectin 5A, in cui il gruppo acetile è alloggiato all’interno di una
cavità idrofobica sulla superficie della proteina mentre il resto della molecola
risulta esposto al solvente.
Altre due forme cristalline della proteina nativa, un’altra ortorombica ed una
trigonale, sono state ottenute e risolte con il metodo della sostituzione molecolare
utilizzando le coordinate di un monomero. La seconda forma cristallina
ortorombica cresce nelle stesse condizioni della prima ed è molto probabilmente il
risultato di un riarrangiamento dell’impacchettamento cristallino di quest’ultima:
l’unità asimmetrica è infatti praticamente identica e la cella unitaria differisce solo
per la minore lunghezza dell’asse c. La terza forma cristallina, invece, appartiene
al gruppo spaziale trigonale P3 ed è stata ottenuta con una diversa soluzione
cristallizzante. L’unità asimmetrica è un esamero formato da tre dimeri. L’analisi
dei contatti intermolecolari e dell’ orientazione dei monomeri in ciascun dimero
dimostra che ciascuno di questi è molto simile a quelli che costituiscono l’unità
asimmetrica delle forme cristalline ortorombiche. La presenza della medesima
unità strutturale in diverse forme cristalline è stata interpretata come un’ ulteriore
prova a favore della natura dimerica di questa lectina.A novel lectin present in carp eggs was recently discovered and
characterized in our laboratory and named Fish Egg Lectin (FEL). FEL is a
glycoprotein of 238 amino acids with a molecular weight of 26.6 kD. The sequence
analysis showed a significant similarity with a lectin present in the plasma and
haemocytes of the horseshoe crab Tachypleus tridentatus, Tachylectin 1, involved
in the non specific immune response of the invertebrate. Biochemical studies
performed by the research group of Prof. Galliano (University of Pavia) have
evidenced that this lectin is present in solution as a monomer and can bind to
saccharides on the surfaces of Gram positive and negative bacteria. N-
acetylglucosamine and N-acetylgalactosamine show the highest affinity among the
monosaccharides while the lectin has little or no affinity for simple sugars or
disaccharides as lactose. Immunohistochemical studies demonstrated that FEL is
an egg specific protein and that it is absent from every other tissue in fish. It is the first time that a lectin like this is described in a vertebrate and its real biological
role has still to be understood.
This thesis focuses on the three-dimensional structure study of FEL by X-
ray diffraction analysis of single crystals. After numerous trials, diffraction quality
crystals of the native protein were prepared using a solution of 15% 2-propanol,
20% PEG 6000, 0.1 M HEPES pH 7.5 as precipitant. They are orthorhombic and
belong to the space group P212121, with cell parameters a = 44.72 Å, b = 72.28 Å
and c = 167.59 Å.
The phase problem was solved by the multiple isomorphous replacement
method. Several derivatives were prepared by soaking protein crystals in mother
liquor containing the heavy atom compound and the diffraction data were collected
and analysed. The best three derivatives (K2PtCl4, Hg(Ac)2 and UO2(Ac)2) were
used in the phase determination process. 2.5 Å resolution electron density maps
were calculated and the molecular model was built and refined.
In the asymmetric unit this lectin is present as a homodimer in which both
monomers have a six β-propeller fold, a highly symmetric structure made up six
“blades” of antiparallel β-strands that surround a central cavity. The analysis of
intermolecular contacts and the change in accessible surface area upon dimer
formation indicate that the dimeric form of FEL is the real physiological unit and
not an artefact of crystal packing. A calcium-binding site was identified and
described in the central tunnel of each monomer.
The binding of monosaccharides was investigated by soaking FEL crystals
in mother liquor containing 0.1 M N-acetylglucosamine and collecting the
diffraction data of the complex. Only one GlcNAc binding site was identified in one
of the monomers, in contrast with the presence of multiple binding sites described
for other β-propeller lectins. The evidence of a single site, especially in a
homodimer, is more likely due to the method used for complex preparation or to
the low affinity for the ligand rather than to a real peculiarity of this protein. The
binding of GlcNAc resembles that described for another lectin of Tachypleus
tridentatus, Tachylectin 5A, with the acetyl group located in a hydrophobic cavity of
the molecular surface and the glycosidic ring exposed to the solvent.
Two other crystal forms were obtained and solved by molecular
replacement with the coordinates of a monomer as search probe. A second
orthorhombic form grows more slowly in the same conditions of the first one and it
can be considered the result of a rearrangement of the crystal packing: the
asymmetric unit is basically identical in the two forms and the unit cell differs only
for the shorter length of the c axis.
The third crystal form is trigonal and belongs to the space group P3. It was
obtained under different crystallization conditions and the asymmetric unit is a
hexamer arranged as a trimer of dimers. The analysis of the intermolecular
contacts and the orientation of the monomers in each dimer revealed that they are
very similar to each other and to those that make up the asymmetric unit of the
orthorhombic forms. The presence of the same structural unit in three different
crystal forms reinforces the assumption of the physiological dimeric nature of this
lectin
BEL -trefoil: A novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms
Full text linkA novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as β-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) β-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galβ1-3GalNAc, was examined in detail. All the three potential binding sites present in the β-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.Fil: Bovi, Michele. Universita Di Verona; ItaliaFil: Cenci, Lucia. Universita Di Verona; ItaliaFil: Perduca, Massimiliano. Universita Di Verona; ItaliaFil: Capaldi, Stefano. Universita Di Verona; ItaliaFil: Carrizo Garcia, Maria Elena. Universidad Catolica de Córdoba. Facultad de Medicina. Departamento de Química Biologica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Civiero, Laura. Università di Padova; ItaliaFil: Chiarelli, Laurent R.. Università di Pavia; ItaliaFil: Galliano, Monica. Università di Pavia; ItaliaFil: Monaco, Hugo L.. Universita Di Verona; Itali
Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective
No full textIn recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms
Untangling the complexity and impact of tau protein ubiquitination
No full textThe microtubule-associated protein tau is an intrinsically disordered protein highly expressed in neuronal axons. Inhealthy neurons, tau regulates microtubule dynamics and neurite outgrowth. However, pathological conditions cantrigger aberrant tau aggregation into insoluble filaments, a hallmark of neurodegenerative disorders known astauopathies. Tau undergoes diverse posttranslational modifications (PTMs), suggesting complex regulation andpotentially varied functions. Among PTMs, the role and mechanisms of ubiquitination in physiology and disease haveremained enigmatic. The past three decades have witnessed the emergence of key studies on tau protein ubiquitination.In this concept, we discuss how these investigations have begun to shed light on the ubiquitination patterns ofphysiological and pathological tau, the responsible enzymatic machinery, and the influence of ubiquitination on tauaggregation. We also provide an overview of the semi-synthetic methods that have enabled in vitro investigations ofconformational transitions of tau induced by ubiquitin modification. Finally, we discuss future perspectives in the fieldnecessary to elucidate the molecular mechanisms of tau ubiquitination and clearance
Structural characterization of two bile acid-binding proteins in zebrafish (Danio rerio)
No full textStructural characterization of two bile acid-binding proteins in zebrafish (Danio rerio
Structural characterization and interaction studies of humanlipocalin-type prostaglandin D synthase (L-PGDS)
Full text linkLipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerisation of the 9,11-endoperoxide group of PGH2 (Prostaglandin H2) to produce PGD2 (Prostaglandin D2) with 9-hydroxy and 11-keto groups in the presence of sulphydryl compounds. PGH2 is a common precursor of all prostanoids, which include thromboxanes, prostacyclins and prostaglandins. PGD2 is synthesized in both the central and peripheral nervous system and it is involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as β-trace protein, the second most abundant protein in human cerebro-spinal fluid. L-PGDS is also detected in brain, testis and prostate, endothelial cells, placenta and heart tissue and even in macrophages infiltrated in atherosclerotic plaques. In these tissues it participates in many physiological activities as well as in the response to diseases. Currently the main structural and biochemical studies, present in the literature, concern recombinant rat and mouse L-PGDS. In this work we use recombinant human L-PGDS in order to solve its three-dimensional structure by X-ray diffraction and test its affinity for several ligands using Surface Plasmon Resonance (SPR). Wild type human L-PGDS and three mutants (C65A; C65A-K59A; C89/186A) were expressed using E. coli cell strains and subsequently purified by a chitin affinity column, size exclusion and hydrophobic interaction chromatography. Large and highly ordered crystals were used to collect X-ray diffraction data using either a rotating-anode generator or a synchrotron source. The multiple isomorphous replacement method was used to solve the phase problem. In the electron density maps an unidentified density was observed apparently interacting with lysine 59 inside the L-PGDS-C65A cavity; the foreign molecule is probably PEG, an additive present in the crystallization liquors. This hypothesis is supported by the fact that the L-PGDS-C65A/K59A crystals, which grow without PEG, show a completely free protein cavity. A seeding experiment of L-PGDS-C65A/K59A crystal, grown in L-PGDS-C65A crystallization conditions, partially confirmed this hypothesis since the foreign molecule was present in the L-PGDS-C65A/K59A cavity. Another crystal form was obtained by mixing L-PGDS-C65A/K59A with the amyloid β peptide (1-40). Although the amyloid β peptide is not visible in the maps, the packing of the protein molecules has changed in the presence of the peptide suggesting interaction of the two molecules. Wild type L-PGDS small crystals were recently obtained and will be tested as soon beam time at a synchrotron source becomes available.
SPR experiments are also in progress and will be used to verify interaction of L-PGDS with PEG, the amyloid β peptide and other ligands and to determine their binding constants
STRUCTURAL CHARACTERIZATION OF HUMAN LIPOCALIN-TYPEPROSTAGLANDIN D SYNTHASE (L-PGDS)
No full textLipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerisation of the 9,11-endoperoxide group of PGH2 (Prostaglandin H2) to produce PGD2 (Prostaglandin D2) with 9-hydroxy and 11-keto groups in the presence of sulphydryl compounds. PGH2 is a common precurson of all prostanoids, which include thromboxanes, prostacyclins and prostaglandins. PGD2 is synthesized in both the central nervous system and the peripheral tissues where it is involved in the maintenance of the body temperature, regulation of nerve cell function, regulation of the sleep wake cycle, tactile pain sensitivity, allergic asthma, inhibition of platelet aggregation and chemotactic recruitment of inflammatory cells. L-PGDS belongs to the lipocalin family and it is able to bind and transport small hydrophobic molecules; it is also the first member of this important family to be recognized as an enzyme. Recently L-PGDS was identified to be already known as the β-trace protein, which is the second most abundant protein in human cerebro-spinal fluid. L-PGDS is also detected in brain, human testis and prostate, endothelial cells, placenta cells, heart tissue and even in macrophages infiltrated to atherosclerotic plaques. In those tissues L-PGDS is involved in many physiological activities as well as in the response to diseases such as diabetes, cardiovascular lesions, multiple sclerosis, Alzheimer’s disease and tumors. Currently the main structural and biochemical studies, present in the literature, concern recombinant rat and mouse L-PGDS. The aim of this work was to express, purify and crystallize recombinant human L-PGDS in order to solve its three-dimensional structure by X-ray diffraction experiments. Wild type human L-PGDS and three mutants (C65A; C65A-K59A; C89/186A) were expressed using E. coli cell strains and subsequently purified by a chitin affinity column, size exclusion chromatography and hydrophobic interaction chromatography. The purification method was improved to obtain highly homogeneous protein suitable for preliminary crystallization trials. Crystallization conditions were optimized to obtain large and highly ordered crystals that were tested by X-ray diffraction using either a rotating-anode generator or a synchrotron source. The multiple isomorphous replacement technique was used to solve the phase problem and heavy atom derivatives were obtained by soaking. An unidentified electron density was observed that seemed to interact with lysine 59 inside the L-PGDS-C65A cavity. It was not possible, at that moment, to characterize this residual molecule although different protocols were tested, including the use of physiological ligands. The L-PGDS-C65A/K59A structure showed a completely free cavity. It was noticed that L-PGDS-C65A/K59A crystals grew without PEG as precipitant, which instead was necessary for the L-PGDS-C65A crystals, suggesting that PEG could be the foreign molecule. A seeding experiment of L-PGDS-C65A/K59A crystal, grown in L-PGDS-C65A crystallization conditions, partially confirmed this hypothesis since the foreign molecule was present in the L-PGDS-C65A/K59A cavity. A high resolution data set of a L-PGDS-C65A gave us the possibility to well define the boundaries of the unknown electron density showing that the foreign molecule was probably PEG. An important crystal structure was obtained by mixing L-PGDS-C65A/K59A with the amyloid β peptide (1-40). Although structure refinement is work in progress, it was the first structural evidence of the interaction of L-PGDS with the amyloid β peptide (1-40). Wild type L-PGDS and L-PGDS C89/186A were purified but they were not homogeneous and no crystals grew in any of the crystallization trials
X-ray evidence of a native state with increased compactness populated by tryptophan-less B. licheniformis β-lactamase.
No full textβ-lactamases confer antibiotic resistance, one of the most serious world-wide health problems, and are an excellent theoretical and experimental model in the study of protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class-A β-lactamase with three tryptophan residues located in the protein core. Here, we report the 1.7-Å resolution X-ray structure, catalytic parameters, and thermodynamic stability of ESP(ΔW), an engineered mutant of ESP in which phenylalanine replaces the wild-type tryptophan residues. The structure revealed no qualitative conformational changes compared with thirteen previously reported structures of B. licheniformis β-lactamases (RMSD = 0.4-1.2 Å). However, a closer scrutiny showed that the mutations result in an overall more compact structure, with most atoms shifted toward the geometric center of the molecule. Thus, ESP(ΔW) has a significantly smaller radius of gyration (R(g)) than the other B. licheniformis β-lactamases characterized so far. Indeed, ESP(ΔW) has the smallest R(g) among 126 Class-A β-lactamases in the Protein Data Bank (PDB). Other measures of compactness, like the number of atoms in fixed volumes and the number and average of noncovalent distances, confirmed the effect. ESP(ΔW) proves that the compactness of the native state can be enhanced by protein engineering and establishes a new lower limit to the compactness of the Class-A β-lactamase fold. As the condensation achieved by the native state is a paramount notion in protein folding, this result may contribute to a better understanding of how the sequence determines the conformational variability and thermodynamic stability of a given fold
Expression, purification and crystallization attempts of Human Bile Acid-CoA:Amino acid N-Acyltransferase (BAAT)
No full textExpression, purification and crystallization attempts of Human Bile Acid-CoA:Amino acid N-Acyltransferase (BAAT
- …
