Littérature scientifique sur le sujet « Biocrystallography »

New academic courses for teaching protein crystallography to biology and chemistry students have been developed. The general aim of the lecture courses is to introduce crystallographic terminology and modern diffraction methodology, to discuss the principles of macromolecular structure, to develop confidence in assessing macromolecular models, and to develop skills in extracting biostructural information from crystallographic literature as well as from bioinformatics resources available on the Internet. Emphasis on structural biology (chemists) or on crystallographic concepts and methodology (biologists) differs depending on the background of the students. In practical classes, the students work with real models of crystals and lattices, and use construction kits to build models of protein secondary structure. Diffraction images of oriented protein crystals are used for determination of unit-cell parameters. An Internet-based multiple-choice quiz is used for tests, self-assessment and practice.

Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine `non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.

The term “crystallogenesis” was primarily mentioned in 1730. I. Newton in his research “Optics”described a phenomenon of regular structure formation from salt solutions. The latter proved to be the origin of nowadays biocrystallography. Later crystallography has been also applied in pharmacy (medication synthesis) and forensic medicine (toxicology). Moreover, a number of clinically oriented works on crystallography have been issued. This method is simple and safe for investigated people and animals. The purpose of this new medico-biological science is to discover crystallogenesis mechanisms and further to work out the criteria for estimation of various substrates bioinformation and biocrystallisation management on the basis of up-to-date accomplishments in the homoeopathy.

Modern spectroscopic techniques grant various methods for a protein structure determination among with a ligand interaction. This work aims at probing the structural insights of a protein-small molecule interaction with biocrystallography and optical spectroscopies. Two independent systems were investigated in the frame of this thesis. The first one involves flavoenzyme interaction with a natural nucleotide as a cofactor required for its catalytic activity and work was purely based on macromolecular crystallography. The second concerns incorporation of a synthetic fluorescent ligand into a model protein as a solution for hydrophobicity of the probe. Due to the nature of the probe optical spectroscopies (such as absorption, fluorescence lifetime, circular dichroism) were effectively employed together with the crystallographic methodology.

L'obiettivo di questo lavoro di tesi era determinare la struttura tridimensionale di tre proteine umane, Heat shock protein 60 (HSP60), Activation-induced cytidine deaminase (AICDA) e Cholesterol 7-alpha-monooxygenase (CYP7A1), attraverso la tecnica di diffrazione di raggi X. La chaperonina umana HSP60 è una proteina mitocondriale, espressa in maniera costitutiva. La proteina è stata espressa in E. coli e purificata tramite cromatografia di affinità immobilizzata dello ione metallo, utilizzando un tag di sei istidine inserito all'estremità N-terminale della proteina, e tramite cromatografia a esclusione molecolare. Le prove di cristallizzazione non danno risultati positivi, probabilmente per problemi di eterogeneità della proteina. La AICDA umana è una citidina deaminasi, la quale è selettivamente espressa nei linfociti B e svolge un ruolo cruciale nell'ipermutazione somatica e nella ricombinazione per scambio di classe degli anticorpi. L'espressione della proteina è stata tentata in E. coli con differenti vettori ed è stata ottenuta con successo con il plasmide pGEX-4T-1, che permette di esprimere la GST all'estremità N terminale della proteina oggetto di studio; sfortunatamente, il protocollo di purificazione non è stato efficace. La CYP7A1 è un enzima microsomale che catalizza la conversione del colesterolo a 7α-idrossicolesterolo, la prima reazione della sintesi degli acidi biliari, e questa è la reazione limitante la velocità della via metabolica; la proteina è espressa solamente nel fegato. L'espressione della proteina umana CYP7A1 in E. coli è stata difficoltosa e, sebbene siano stati testati differenti vettori, non è stato ottenuto un buon livello di espressione della proteina. Per superare questi problemi, la CYP7A1 di pesce zebra (Danio rerio) è stata espressa in E. coli, ma senza apprezzabili miglioramenti. Un'altra parte della mia tesi ha riguardato la determinazione della struttura tridimensionale della Methionine aminopeptidase 1 umana con due differenti inibitori. La proteina catalizza l'eliminazione della metionina N-terminale dalle proteine nascenti. Inibitori contro questa proteina sono di grande interesse medico grazie al loro potenziale impiego come farmaci antitumorali. Questa parte di tesi è stata svolta presso il laboratorio del Dr. L. Mario Amzel sotto la supervisione della Dr. Sandra B. Gabelli - Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine (Baltimore, USA).
The aim of this thesis work was to determine the three-dimensional structure of three human proteins, Heat shock protein 60 (HSP60), Activation-induced cytidine deaminase (AICDA) and Cholesterol 7-alpha-monooxygenase (CYP7A1), by X-ray diffraction of single crystals. The human chaperonine HSP60 is a mitochondrial protein, expressed in a constitutive manner. The protein was expressed in E. coli and purified by immobilized metal ion affinity chromatography, using a histidine tag fused to the N-terminus of the protein, and by size exclusion chromatography. The crystallization trials do not give positive results, probably due to protein heterogeneity problems. Human AICDA is a cytidine deaminase, which is selectively expressed in B lymphocytes and plays a crucial role in antibody somatic hypermutation and class switch recombination. Protein expression was attempted in E. coli with different vectors and was successfully achieved with the pGEX-4T-1 plasmid, that allows to express GST at the N-terminus of the target protein; unfortunately, the purification protocol was not effective. CYP7A1 is a microsomal enzyme that catalyzes the conversion of cholesterol to 7α-hydroxycholesterol, the first reaction of bile acid synthesis and the rate-limiting step of the metabolic pathway; the protein is expressed only in the liver. The expression of human CYP7A1 in E. coli was troublesome and, although different vectors were tested, a good level of protein expression was not obtained. In order to overcome these problems, zebrafish (Danio rerio) CYP7A1 was expressed in E. coli, but without any substantial improvement. Another part of this thesis work concerned the determination of the three-dimensional structure of the human Methionine aminopeptidase 1 with two different inhibitors. The protein catalyzes the removal of the N-terminal methionine from nascent proteins. Inhibitors against this protein are of great medical interest because of their potential employment as anticancer drugs. This part of the thesis was performed at Dr. L. Mario Amzel's laboratory under the direct supervision of Dr. Sandra B. Gabelli - Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine (Baltimore, USA).

Questo lavoro di tesi riguarda la purificazione e la caratterizzazione strutturale di una nuova lectina da funghi porcini. Questa lectina è stata chiamata BEL (Boletus edulis lectin) beta-trefoil e presenta un fold totalmente diverso da quello descritto per le lectine da fungo solubili in soluzione fisiologica. La BEL beta-trefoil è stata studiata approfonditamente dal punto di vista strutturale, determinandone sia la sequenza aminoacidica che la struttura tridimensionale. Questa nuova proteina è costituita da un omodimero in cui ogni monomero è ripiegato secondo il ben noto beta-trefoil fold come si evince dal nome proposto per essa. La lectina è dotata di un evidente effetto antiproliferativo nei confronti di cellule cancerogene umane e questo le conferisce un potenziale terapeutico nella cura dei carcinomi. Per quanto riguarda lo studio strutturale sono state analizzate, mediante diffrazione di raggi X, diverse forme cristalline della proteina in forma apo e numerosi complessi. La migliore forma cristallina della proteina apo raggiunge un limite di risoluzione di 1.12 Å. È stata inoltre analizzata in maniera dettagliata l’interazione della lectina con alcuni carboidrati: lattosio, galattosio, N-acetilgalattosammina e il T-Antigen (Ser-Gal beta1-3GalNAc), il disaccaride responsabile dell’interazione della lectina con le cellule tumorali. La lectina presenta tre siti di legame per gli zuccheri e tutti sono occupati in almeno una forma cristallina. Infine confrontando la struttura della proteina apo con quella dei complessi con gli zuccheri non sono stati osservati cambiamenti conformazionali rilevanti.
This thesis deals with the purification and the structural characterization of a novel lectin, completely different from the formerly described members of the saline soluble family of mushroom specific lectins. The lectin was called BEL (Boletus edulis lectin) beta-trefoil and it was purified from the fruiting bodies of king bolete mushrooms, commonly known as porcini. BEL beta-trefoil was structurally characterized: its amino acid sequence and three dimensional structure were determined. The new protein is a homodimer and each protomer folds as a beta-trefoil domain as indicated by the name itself. 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, the T-antigen disaccharide (Gal beta1-3GalNAc) and the T-Antigen (Ser-Gal beta1-3GalNAc) was examined in detail. All the three potential binding sites present in the beta-trefoil fold are occupied in at least one crystal form. No important conformational changes are observed in the lectin when comparing the structure of the complexes with carbohydrates and those of the ligand free protein.