1,721,044 research outputs found
What proteomics can do for cultural heritage
No full textWHAT PROTEOMICS CAN DO FOR CULTURAL HERITAGE?
Gabriella Leo, Piero Pucci, Gennaro Marino, Leila Birolo.
Dipartimento di Chimica Organica e Biochimica, Università di Napoli “Federico II”, Napoli, Italy.
In the context of artistic and historic objects, the identification of proteins is still a challenging task, because of the very low amount of sample available, because of the complex and quite variable chemical composition of the paints, because of the possible simultaneous presence of several components, and because of degradation of the original materials as a result of aging and pollution. We proposed to adapt proteomic strategies for the identification of proteins in binders of paintings, but also in seeds, food remains in archeological find, etc.., to overcome requirements and difficulties presented by specific samples. In particular, tryptic hydrolysis in heterogeneous phase, followed by the analysis by LC-MS/MS, was successfully used to unambiguously identify milk proteins in a sample from a painting decorating the vaults of the upper church in the Basilica of St. Francis in Assisi (Leo et al., 2009).
Moreover, similar strategies were exploited for the characterization of the natural and unnatural aging products in masterpieces. We identified deamidation as a major modifications of proteins in binders, that could be related to the aging of artistic and historic objects (Leo et al., in preparation).
The systematic analysis of samples from the 14th century frescoes of the Monumental Cemetery in Pisa indicate extensive deamidation occurring on most of the peptides identified, suggesting that the miniature molecular clock, as Robinson and Robinson defined any amide residue present in peptides or proteins (2004), might well be used as molecular marker in artworks
PROTEOMICS FOR THE DIAGNOSIS OF DETERIORATION OF WALL PAINTINGS
No full textPROTEOMICS FOR THE DIAGNOSIS OF DETERIORATION OF WALL PAINTINGS
Gabriella Leo1, Piero Pucci1, Gennaro Marino1, Leila Birolo1. 1Dipartimento di Chimica Organica e Biochimica, Università di Napoli “Federico II”, Napoli, Italy.
Ilaria Bonaduce2, Alessia Andreotti2, Maria Perla Colombini2. 2Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Italy.
The pysico-chemical analysis of wall paintings is particularly challenging due to the vast range of inorganic and organic materials which often constitute them. Although the organic paint media can be often reliably identified, changes in the composition, degradation phenomena, the formation of minor components, and molecular modifications that have occurred as an effect of production processes and aging cannot be completely understood by using only gas chromatography/mass spectrometric based approaches. In these cases the support of other techniques able to give information also from the macromolecular composition point of view are often fundamental and, up to now, they have been used for limited number of cases. The application of proteomics strategy to the characterization of proteinaceous binders in paintings has been recently reported in the literature. Actually, these studies were dedicated to setting up a protocol to be used to identify proteinaceous paint media, and not focused to a better understanding of modifications and degradation phenomena undergone as an effect of processing treatments used by artists and restorers, as well as ageing. This paper presents a proteomic approach able to characterize the proteinaceous material and to evaluate the degradation pathway occurred to the wall painting. Particularly, a systematic analysis of samples from the 14th century frescoes of the Monumental Cemetery in Pisa indicates an extensive deamidation occurring on most of the peptides identified, suggesting that the miniature molecular clock, as Robinson and Robinson defined any amide residue present in peptides or proteins, might well be used as molecular marker in artworks. Deamidation, the major modification of casein and animal glue proteins present in the wall paintings, can be related to treatments undergone by the proteinaceous materials during the after war restoration, as well as due to aging effects induced by the microclimate of the Monumental Cemeter
Studio delle interazioni tra proteine amiloidogeniche e ligandi mediante spettrometria di massa
No full textAnalisi conformazionali di mutanti della beta-2-microglobulina e loro interazione con il collageno
No full textProtein-protein interactions: characterization and new strategy for industrial applications
No full textBiological systems are made up of very large numbers of different components
interacting at various scales. Most genes, proteins and other cell components carry
out their functions within a complex network of interactions and a single component
can affect a wide range of other components. Interactions involved in biological
processes have been previously characterized individually, but this ‘‘reductionist’’
approach suffers from a lack of information about time, space, and context in which
the interactions occur in vivo. A global, integrative, approach has been developed for
several years, focusing on the building of protein-protein interaction maps
(interactomes). These interaction networks are complex systems, where new
properties arise. This is part of an emergent field, called systems biology which is
‘‘the study of an organism, viewed as an integrated and interacting network of genes,
proteins and biochemical reactions which give rise to life’’
(http://www.systemsbiology.org/). This interdisciplinary approach, involving
techniques from the mathematical, computational, physical and engineering sciences
is required to understand complex networks. Detailed knowledge of protein
interactions and protein complex structures is therefore fundamental to understand
how individual proteins function within a complex and how the complex functions as a
whole.
This PhD thesis targeted the characterization by structural and functional proteomics
approaches of selected protein complexes involved in relevant molecular processes
and can have an impact on their industrial applications. The following systems of
biotechnological interest have been analysed in this study: 1) Phenol Hydroxylase
(PH) from Pseudomonas sp. OX1. It is a bacterium endowed with the ability to grow
on a wide spectrum of hydroxylated and non-hydroxylated aromatic compounds for
this reason it can be use in bioremediation of contaminated environments. In this
context the research has been aimed at characterizing the endogenously expressed
Phenol Hydroxylase (PH) complex from Pseudomonas sp. OX1, using biochemical
approaches integrated to mass spectrometry analysis. Moreover, in order to define
the protein-protein interaction networks of this complex a functional proteomic
approach was carried out. 2) Characterization of the complex between pLG72
and human D-amino acid oxidase (hDAAO), linked to the onset of
schizophrenia. The molecular basis of schizophrenia is still elusive and current
treatments focus on eliminating the symptoms of the disease. The clarification of the
role of this specific complex and the identification of other physiological or conditional
partners in this biochemical process will be useful in developing molecules that can
be effective in the disease treatment.
Moreover, as a parallel goal, and strictly connected with the former issue, I worked at
3) the development of an innovative structural analysis approach based on the
use of a femtosecond UV laser as a novel zero length protein-protein crosslinker.
Such photo-physical approach could obviate many of the problems
associated with standard chemical cross-linking reagents, and put cross-linking in a
proteome-wide position for the characterizations of protein-protein interactions in vivo
in intact cells, the study of the transient interactions among proteins, mapping
molecular interfaces in protein complexes, providing information on the dynamics of
the contacts within a multi-protein complex
Spettrometria di massa
No full textLa sperimentazione e la ricerca di materiali sempre nuovi che garantissero risultati ottimali e fossero stabili nel tempo è sempre stata, sin dall’antichità, un aspetto estremamente importante dell’attività artistica. Questa “ricerca sperimentale” degli artisti costituisce un importante, stimolante ed impegnativo scoglio per il lavoro dei restauratori, ulteriormente complicato da precedenti interventi di restauro che a loro volta hanno inserito ulteriori modifiche nei materiali delle opere d’arte. Una rapida scorsa alla recente letteratura sull’analisi e caratterizzazione dei materiali organici utilizzati in opere d’arte della più svariata provenienza, mostra chiaramente come le tecniche di spettrometria di massa, accoppiate o meno a tecniche di separazione cromatografica, assumano sempre più un ruolo centrale e determinante
The Roles of Tyr70 and Tyr225 in Aspartate Aminotransferase Assessed by Analysing the Effects of Mutations on the Multiple Reactions of the Substrate Analogue Serine O-Sulphate
No full textAspartate aminotransferase catalyses multiple reactions of the glutamate analogue, serine O-sulphate. The predominant reaction is beta-elimination of sulphate to give aminoacrylate (k(cat) = 13 s(-1) for the Escherichia coli enzyme) which may either hydrolyse to pyruvate and ammonia, or react covalently with the enzyme and inactivate it (k(inact) = 1.1x10(-3) s(-1)). Serine O-sulphate also undergoes a transamination reaction that converts the enzyme to its pyridoxamine form (k(cat) = 0.11 s(-1)). Tyr70 and Tyr225, each of which forms a hydrogen bond with the coenzyme, were substituted with methionine and phenylalanine, respectively. The Y225F mutation does not affect beta-elimination but reduces the rates of transamination and inactivation about 70-fold and 3-fold, respectively. Apparently, Tyr225 is not essential for the steps leading to and including abstraction of the proton from C alpha of the substrate. It is argued that the Y225F mutation interferes with ketimine hydrolysis The Y70M mutation affects all three reactions, beta-elimination being about fourfold slower, transamination 340-fold slower, and inactivation being 1.4 times faster than in the wild-type enzyme. It is proposed that a hydrogen bond from Tyr70 positions Lys258 for protonation of the quinonoid intermediate at C4' and that, although the full kinetic contribution of this interaction is only revealed in the multiple reactions of serine O-sulphate, the same interaction is equally important in increasing the reaction specificity for transamination of the natural substrates
Structural characterization of the M* partly folded intermediate of wild-type and P138A EcAspAT
Full text linkA combination of spectroscopic techniques, hydrogen/deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M*partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an on-pathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M*intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N2 forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposabl
Characterization of the human D-amino acid oxidase (hDAAO) - pLG72 complex involved in the onset of schizophrenia.
No full textSchizophrenia is a chronic and severely debilitating psychiatric disorder affecting nearly 1% of the world’s population. In 2002, the new human gene G72, encoding for the pLG72 protein, and the gene encoding for DAAO have been genetically linked to the susceptibility to schizophrenia. A yeast two-hybrid screening experiment identified D-amino acid oxidase (DAAO) as a putative interacting partner of pLG72. DAAO is a FAD-containing flavooxidase that in brain is responsible for the elimination of D-serine, a co-agonist that binds to the glycine-site of the NMDA receptor. We recently demonstrated that pLG72 acts as “inactivator” of human DAAO and that the cellular concentration of D-serine depends on the expression of the active form of this flavooxidase. Based on these results, a molecular model for the onset of schizophrenia has been proposed: a decrease in pLG72 expression might yield an anomalous high level of hDAAO activity and therefore a decrease in the local concentration of D-serine, affecting glutamatergic neurotransmission mediated by NMDA receptor.
The characterization of the complex is a challenging task, hardly feasible by high resolution techniques, since: 1) structural informations on pLG72 are lacking; 2) pLG72 is soluble only in the presence of mild denaturant; 3) no homologous protein has been structurally characterized so far. In this perspective, we have used low resolution strategies based on the coupling of classical biochemistry approaches (complementary proteolysis, cross-link) with mass spectrometric techniques, to characterize the pLG72-hDAAO complex. Results indicated that hDAAO exhibits different proteolysis profiles when isolated or in complex with pLG72, thus suggesting a conformational change upon binding the effector protein. Chemical cross-linking experiments will complement the proteolysis experiments providing with details the contact regions between hDAAO e pLG72
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