17 research outputs found
Crystallographic analysis of structure and function of cyclophilin enzymes and study of inclusion compounds of natural products in cyclodextrins
No full textIn this thesis the X-ray crystallographic analysis of large and medium-small size biomolecules and their complexes is presented. The aim of the first part is the crystal structure determination of the cytoplasmic cyclophilin-A from Azotobacter vinelandii (AvCyPA or AvPPIB) bacterium. Cyclophilins catalyze the cis-trans isomerization of peptide bonds preceding proline residues of protein substrates. The crystal structure of AvCyPA was determined at 2.2 Å resolution. In addition, the crystal structure of the enzyme complexed with the synthetic tetrapeptide succinyl-Ala-Phe-Pro-Phe-p-nitroanilide was determined at 2.0 Å resolution and shows that the tetrapeptide proline adopts a cis-isomer conformation. Morever, we designed point mutants by changing aminoacids that are located outside from the active site of the enzyme in order to examine the influence of such a substitution on the overall dynamic structural network involved in catalysis. The AvPPIBA84S, AvPPIBA84T and AvPPIBM49A mutants showed little or no difference in PPIase activity in comparison with the native enzyme. The second part of this thesis deals with the crystal structures of the inclusion compounds of (d)- and (l)-borneol enantiomers in α- and β-cyclodextrin by X-ray crystallography. Borneol is a bicyclic monoterpene present in the essential oils of numerous medicinal plants used in pharmaceutical industry. Modified cyclodextrins have been used for chiral recognition of the two enantiomeric forms. The crystallographic study of these inclusion compounds aims to illustrate borneol enantioselectivity by complexation in native cyclodextrins. The crystal structures of the inclusion compounds of (d)- and (l)-borneol enantiomers in α- and β-CD were determined by X-ray crystallography at about 1 Å resolution. The complexes of the borneol enantiomers with the same host molecule have the same inclusion geometry. The results of this crystallographic analysis have shown that the borneol molecule is found significantly less disordered within the α-CDs dimer cavity than the larger β-CDs dimer cavity indicating that the complexation of the borneol with α-CD is more stable compared to that with β-CD and the encapsulated d-borneol molecule in the α- or β-CD dimer is found significantly less disordered than the l-borneol in the corresponding complexes.Στη διατριβή αυτή, παρουσιάζεται η κρυσταλλογραφική ανάλυση με ακτίνες-Χ μεγάλων και μικρο-μεσαίων βιομορίων και συμπλόκων τους. Σκοπός του πρώτου μέρους της διατριβής είναι η επίλυση της δομής του ενζύμου της κυτταροπλασματικής κυκλοφιλίνης-Α από το αζωτοβακτήριο Azotobacter vinelandii (AvCyPA ή AvPPIB). Οι κυκλοφιλίνες καταλύουν την cis-trans ισομερίωση των πεπτιδικών δεσμών που προηγούνται του κατάλοιπου της προλίνης σε πρωτεϊνικά υποστρώματα. Η κρυσταλλική δομή του ενζύμου της AvCyPA προσδιορίστηκε σε διακριτότητα 2.2 Å, ενώ η κρυσταλλική δομή της σε σύμπλοκο με το συνθετικό τετραπεπτίδιο ηλεκτρύλιο– Ala–Phe–Pro–Phe–p–νιτροανιλίδιο σε 2 Å. Η προλίνη του πεπτιδίου υιοθετεί την cis διαμόρφωση. Παράλληλα, σχεδιάστηκαν σημειακές μεταλλάξεις εκτός του ενεργού κέντρου του ενζύμου προκειμένου να μελετηθεί η επίδρασή τους στη δομή και λειτουργία του ενζύμου. Ωστόσο, τα μεταλλάγματα AvPPIBA84S, AvPPIBΑ84Τ και AvPPIBΜ49Α δεν βρέθηκε να παρουσιάζουν σημαντικές διαφορές στην δράση ισομεράσης σε σχέση με την φυσική πρωτεΐνη. Στο δεύτερο μέρος της διατριβής παρουσιάζεται η ανάλυση των προϊόντων εγκλεισμού των d- και l- εναντιομερών βορνεόλης (ξενιζόμενα μόρια) σε α- και β- κυκλοδεξτρίνη (α-, β-CD) (μόρια ξενιστές) με κρυσταλλογραφία ακτίνων-Χ. Η βορνεόλη είναι ένα δικυκλικό μονοτερπένιο, που απαντάται στα αιθέρια έλαια μιας πληθώρας φαρμακευτικών φυτών και χρησιμοποιείται στην φαρμακευτική. Τροποποιημένες κυκλοδεξτρίνες έχουν χρησιμοποιηθεί για τον διαχωρισμού ρακεμικού μίγματος των δυο εναντιομερών. Η δομική μελέτη των συγκεκριμένων προϊόντων εγκλεισμού σε α- και β-CD, σκοπεύει να συμβάλει στην κατανόηση της ικανότητας μοριακής/χειρόμορφης αναγνώρισης από φυσικές κυκλοδεξτρίνες. Οι κρυσταλλικές δομές των προϊόντων εγκλεισμού d-/α-CD, l-/α-CD, d-/β-CD και l-/β-CD επιλύθηκαν με κρυσταλλογραφία ακτίνων-Χ σε διακριτότητα περίπου 1 Å. Η γεωμετρία εγκλεισμού είναι παρόμοια μεταξύ των συμπλόκων των εναντιομερών στον ίδιο ξενιστή. Από την σύγκριση αυτών των δομών προκύπτει ότι το μόριο της βορνεόλης είναι λιγότερο αποδιατεταγμένο όταν εγκλείεται στην μικρότερη κοιλότητα των α-CD και πως η d- βορνεόλη εμφανίζει μικρότερη αποδιάταξη συγκριτικά με την αντίστοιχα εγκλεισμένη l-βορνεόλη
Molecular Symmetry of Permethylated <i>β</i>-Cyclodextrins upon Complexation
No full textThe Cn molecular symmetry implicated by the schemes with which cyclodextrins (CDs), the well-known cyclic oligosaccharides, are introduced in the literature, is not valid. Numerous studies have shown that CDs are rather flexible with their macrocycle adopting various conformations that enable the inclusion complexation of guest molecules of various shapes. In this work, the loss and gain of the C7 symmetry of the heptakis (2, 3, 6-tri-O-methyl)-β-CD (TM-β-CD) is investigated by means of its conformation geometrical features in its hydrated form and upon complexation with molecules of different shapes. For this, the crystal structure of the inclusion complex of a bulky guest molecule (giberellic acid) in TM-β-CD is presented for the first time and compared with the previously determined crystal structures of monohydrated TM-β-CD and the inclusion complex of a linear monoterpenoid (geraniol) in TM-β-CD. The structural investigation was complemented by molecular dynamics simulations in an explicit solvent, based on the crystallographically determined models. The crucial role of the guest, in the symmetry gain of the host, reveals a pronounced induced-fit complexation mechanism for permethylated CDs
Crystal structure of the inclusion complex of cholesterol in β-cyclodextrin and molecular dynamics studies
No full textThe role of beta-cyclodextrin (β-CD) in cholesterol removal primarily from mammalian cells and secondly from dairy products has been studied thoroughly in recent years. Although the physicochemical characterization of the inclusion compound of cholesterol in β-CD has been achieved by various methods, no crystal structure has been determined so far. We report here the crystal structure of the inclusion compound of cholesterol in β-CD. The inclusion complex crystallizes in the triclinic space group P1 forming head-to-head dimers which are stacked along the c-axis. One well-defined cholesterol molecule ‘axially’ encapsulated inside the β-CD dimer and 22 water molecules that stabilize the complexes in the crystalline state comprise the asymmetric unit of the structure. The dimers are arranged in an intermediate (IM) channel packing mode in the crystal. Moreover, MD simulations, at 300 and 340 K, based on the crystallographically determined coordinates of the complex show that the formed cholesterol/β-CD inclusion compound remains very stable in aqueous solution at both temperatures
Molecular Symmetry of Permethylated β-Cyclodextrins upon Complexation
No full textThe Cn molecular symmetry implicated by the schemes with which cyclodextrins (CDs), the well-known cyclic oligosaccharides, are introduced in the literature, is not valid. Numerous studies have shown that CDs are rather flexible with their macrocycle adopting various conformations that enable the inclusion complexation of guest molecules of various shapes. In this work, the loss and gain of the C7 symmetry of the heptakis (2, 3, 6-tri-O-methyl)-β-CD (TM-β-CD) is investigated by means of its conformation geometrical features in its hydrated form and upon complexation with molecules of different shapes. For this, the crystal structure of the inclusion complex of a bulky guest molecule (giberellic acid) in TM-β-CD is presented for the first time and compared with the previously determined crystal structures of monohydrated TM-β-CD and the inclusion complex of a linear monoterpenoid (geraniol) in TM-β-CD. The structural investigation was complemented by molecular dynamics simulations in an explicit solvent, based on the crystallographically determined models. The crucial role of the guest, in the symmetry gain of the host, reveals a pronounced induced-fit complexation mechanism for permethylated CDs
Inclusion Complexes of Naringenin in Dimethylated and Permethylated β-Cyclodextrins: Crystal Structures and Molecular Dynamics Studies
No full textThe crystal structures of the inclusion complexes of naringenin in dimethylated and permethylated β-cyclodextrin (DM-β-CD and TM-β-CD) were determined and extensively analyzed. Naringenin is found with its 4-hydroxyphenyl residue fully immersed in the DM-β-CD cavity and its chromone group protruding from the narrow rim of the open-cone shaped host. The naringenin/DM-β-CD complex units are packed in a ‘herring bone’ fashion. In the case of naringenin/TM-β-CD, the complex units are arranged in a cage-type mode, the guest naringenin is partially encapsulated in the cavity of the closed-cone shaped host, with its chromone group laying equatorially and its 4-hydroxyphenyl protruding extensively from the wide rim of the host. Furthermore, the crystallographically-determined coordinates of both complexes were employed for Molecular Dynaimics simulations in explicit water solvent and in the absence of crystal contacts. The trajectories showed that naringenin rapidly penetrates the open narrow rim of DM-β-CD but not the closed narrow rim of TM-β-CD. Thus, in the latter case, the chromone group of naringenin is accommodated shallowly in the wide rim of the host, tethered via hydrogen bonds to the secondary methoxy groups of the host. Finally, a significantly higher binding affinity for naringenin in DM-β-CD than TM-β-CD was estimated by Molecular Mechanics/Generalized Born Surface Area calculations
Structural and Evolutionary Insights within the Polysaccharide Deacetylase Gene Family of Bacillus anthracis and Bacillus cereus
No full textFunctional and folding constraints impose interdependence between interacting sites along the protein chain that are envisaged through protein sequence evolution. Studying the influence of structure in phylogenetic models requires detailed and reliable structural models. Polysaccharide deacetylases (PDAs), members of the carbohydrate esterase family 4, perform mainly metal-dependent deacetylation of O- or N-acetylated polysaccharides such as peptidoglycan, chitin and acetylxylan through a conserved catalytic core termed the NodB homology domain. Genomes of Bacillus anthracis and its relative Bacillus cereus contain multiple genes of putative or known PDAs. A comparison of the functional domains of the recently determined PDAs from B. anthracis and B. cereus and multiple amino acid and nucleotide sequence alignments and phylogenetic analysis performed on these closely related species showed that there were distinct differences in binding site formation, despite the high conservation on the protein sequence, the folding level and the active site assembly. This may indicate that, subject to biochemical verification, the binding site-forming sequence fragments are under functionally driven evolutionary pressure to accommodate and recognize distinct polysaccharide residues according to cell location, use, or environment. Finally, we discuss the suggestion of the paralogous nature of at least two genes of B. anthracis, ba0330 and ba0331, via specific differences in gene sequence, protein structure, selection pressure and available localization patterns. This study may contribute to understanding the mechanisms under which sequences evolve in their structures and how evolutionary processes enable structural variations
Inclusion Complexes of Naringenin in Dimethylated and Permethylated beta-Cyclodextrins: Crystal Structures and Molecular Dynamics Studies
No full textThe crystal structures of the inclusion complexes of naringenin in
dimethylated and permethylated beta-cyclodextrin (DM-beta-CD and
TM-beta-CD) were determined and extensively analyzed. Naringenin is
found with its 4-hydroxyphenyl residue fully immersed in the DM-beta-CD
cavity and its chromone group protruding from the narrow rim of the
open-cone shaped host. The naringenin/DM-beta-CD complex units are
packed in a `herring bone' fashion. In the case of
naringenin/TM-beta-CD, the complex units are arranged in a cage-type
mode, the guest naringenin is partially encapsulated in the cavity of
the closed-cone shaped host, with its chromone group laying equatorially
and its 4-hydroxyphenyl protruding extensively from the wide rim of the
host. Furthermore, the crystallographically-determined coordinates of
both complexes were employed for Molecular Dynaimics simulations in
explicit water solvent and in the absence of crystal contacts. The
trajectories showed that naringenin rapidly penetrates the open narrow
rim of DM-beta-CD but not the closed narrow rim of TM-beta-CD. Thus, in
the latter case, the chromone group of naringenin is accommodated
shallowly in the wide rim of the host, tethered via hydrogen bonds to
the secondary methoxy groups of the host. Finally, a significantly
higher binding affinity for naringenin in DM-beta-CD than TM-beta-CD was
estimated by Molecular Mechanics/Generalized Born Surface Area
calculations
Structural Studies of Piperine Inclusion Complexes in Native and Derivative β-Cyclodextrins
No full textPiperine (PN), the primary pungent alkaloid in black pepper shows several biological activities such as antioxidant, antimicrobial and anti-cancerogenic effects. Similar to other alkaloids, PN is characterized by poor water solubility. One way to improve its solubility and thus its biological activities is by forming inclusion complexes with suitable cyclodextrins. In this work PN inclusion complexes in native β-cyclodextrin (β-CD), its methylated (randomly methylated (RM-β-CD), heptakis-(2,6-di-O-methyl)-β-CD (DM-β-CD) and heptakis-(2,3,6-tri-O-methyl)-β-CD (TM-β-CD)) and 2-hydroxypropylated (HP-β-CD) derivatives are investigated using physicochemical methods, such as phase solubility study and X-ray crystallography complemented by theoretical (molecular dynamics simulations) studies. The determination of the crystal structure of the PN inclusion complexes in β-CD, DM-β-CD and TM-β-CD, reveals the formation of 1:2 guest:host inclusion complexes in the crystalline state. The guest PN molecule threads the hydrophobic cavities of the hosts which are arranged as couples in a tail-to-tail mode in the case of PN/β-CD and in a head-to-tail mode in the cases of PN/DM-β-CD and PN/TM-β-CD. MD studies based on the crystallographically determined structures and docked models show the stability of the examined complexes in an aqueous environment whereas the binding affinity of PN for the host molecules is calculated by the MM/GBSA method. Finally, phase-solubility studies of PN with β-CD, RM-β-CD and HP-β-CD are presented, indicating a Bs-type for the PN/β-CD complex and an AL-type for the PN/RM-β-CD and PN/HP-β-CD complexes with 1:1 guest:host stoichiometry
X-ray crystallography and molecular dynamics studies of the inclusion complexes of geraniol in β-cyclodextrin, heptakis (2,6-di-O-methyl)-β-cyclodextrin and heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin
No full textThe inclusion of the linear monoterpene, geraniol (gr) in β-cyclodextrin (β-CD), heptakis(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD) and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) has been studied by X-ray crystallography and Molecular Dynamics simulations. The gr/β-CD complex crystallizes as a head-to-head dimer in the P21 space group stacking along the crystallographic a-axis in a channel packing mode. Two guest molecules are accommodated almost axially inside the dimeric cavity with their aliphatic ends laying in the interface region of the dimer facing each other and their hydroxylic ends protruding from the narrow β-CD rims. The guest molecules of the adjacent dimers in a channel are interconnected via CH ⋯O and hydrogen bonds forming an internal wire. Both the gr/DM-β-CD and gr/TM-β-CD complexes crystallize in the P212121 space group and stack along the crystallographic b-axis in a head-to-tail manner. DM-β-CD host adopts the conformation of a rigid and well-shaped open cone upon complexation, in which the guest molecule is found highly disordered over 5 sites with varying depths of immersion. The gr/DM-β-CD complex units are arranged in channels and the gr molecules within these channels are also interconnected via CH ⋯O bonds. On the other hand, TM-β-CD host is found severely distorted adopting a ‘closed’ cup-shaped conformation. The guest is partially encapsulated in the wide rim of TM-β-CD and it is found disordered over 3 sites with quite different orientations. The complex units are arranged forming screw channels, with the main part of the guest laying outside the host cavity and filling the intermediate space between the succeeding hosts. The MD analysis based on the crystallographically determined structures sheds light on the dynamic behavior of the geraniol upon complexation with these hosts, its conformation variations and the interconversion of the inclusion modes in solution. Finally, MM/GBSA-calculations revealed that the ascending order in binding affinity ΔG values is: gr/TM-β-CD < gr/DM-β-CD < gr/β-CD
Glycosidic vs. Aglycol Form of Natural Products as Putative Tyrosinase Inhibitors
No full textNumerous natural products and designed molecules have been evaluated as tyrosinase inhibitors that impede enzymes’ oxidation activity. In the present study, new potent natural inhibitors were retrieved from the ZINC database by the similarity-screening of 37 previously reported tyrosinase inhibitors. The screening resulted in 42 candidate inhibitory molecules that were categorized into five groups. Molecular-docking analysis for these compounds, as well as for three others known for their inhibition activity (caffeic acid, naringenin, and gallic acid), was carried out against the tyrosinase structure from Agaricus bisporus (AbTYR). The top-scoring compounds were used for further comparative analysis with their corresponding naturally occurring glycosides. The results suggested that the glycosylated inhibitors could interact better with the enzyme than their aglycon forms. In order to further examine the role of the sugar side group of potent tyrosinase inhibitors, the dynamic behavior of two such pairs of glycosidic/aglycol forms (naringin–naringenin and icariin–icaritin) in their complexes with the enzyme were studied by means of 20-ns MD simulations. The increased number of intermolecular hydrogen bonds and their augmented lifetime between AbTYR and the glycosidic analogues showed that the naringin and icariin molecules form more stable complexes than naringenin and icaritin with tyrosinase, and thus are more potent inhibitors
