Laboratory of Structural & Supramolecular Chemistry

Research Projects

Cyclodextrin Inclusion Complexes

Synthesis of functional cyclodextrin derivatives

Structure of macromolecules by X-ray crystallography


A. Cyclodextrin Inclusion Complexes: crystalline state and solution structures and properties

Cyclodextrins are natural, water soluble macrocyclic carbohydrate molecules (Fig. A1), oligomers of α-D-glucose, that possess a cavity where a plethora of hydrophobic molecules can be encapsulated. The resulting host-guest inclusion complexes exhibit many characteristics of receptor-substrate systems. The laboratory has a long experience in inclusion complexes of cyclodextrins with various guest systems:


Figure A1. The common cyclodextrins: α-cyclodextrin (αCD), β-cyclodextrin (βCD) and γ-cyclodextrin (γCD). 


I.     Influence of guest on the crystal packing of inclusion complexes

Packing Research on X-ray structures of β-cyclodextrin inclusion complexes with model compounds has revealed that the majority of them forms dimers, which pack in 4 major packing arrangements [1-11]. The hydrophobicity / hydrophilicity of the end groups of the guest that emerge from the primary sides of the host dimer determine the crystal packing. A characteristic example is the difference in packing between the complexes of long aliphatic mono-carboxylic acids and di-carboxylic acids (Fig. A2). 


Figure A2. Inclusion of long mono-carboxylic acids (right, CH packing) and di-carboxylic acids (left, Intermediate packing) into βCD. It is suggested that it is the hydrophobic end-methyl group of the former guests at one of the primary faces of the βCD dimer, what dictates the channel formation in order to shield that part of the guest from the surrounding water molecules. The carboxylic end-group of the guest is found entrapped inside the channel at the other primary face. However, two carboxylic groups from neighboring layers self-associate and form carboxylic dimers, thus stabilizing the whole system. In the case of dicarboxylic acid guests, the end-carboxylic groups are exposed and interact with water molecules.

Of course, all rules have exceptions. Thus flexibility on the part of the guest [1,2-bis(4-aminophenyl)­ethane] that acquires two different conformations in the cavity of the βCD dimer (Fig. A3), results in a packing arrangement different that the four defined above, which however, can be transformed approximately to one of them, the screw channel mode of packing [12 and references therein].


   Figure A3. Inclusion complex βCD/1,2-bis(4-aminophenyl)­ethane.

Another guest, 4-pyridinealdazine,  that has the tendency to form π-π dimers on the one hand, and H-bonds with water molecules on the other, is encapsulated into βCD with the accompanying water molecules and forms βCD trimers (Fig. A4), detected for the first time [13], with host / guest ratio 3:2, a highly unusual stoichiometry. 


Fig. A4. Two βCD trimers forming channels. Each trimer holds a 4-pyridinealdazine dimer (guests in grey) and water clusters (pink) connecting by H-bonds the guests. 

II.     Self-assembly in solution and solid state 

Non-bonding interactions, H-bonding and organization of CD supramolecular systems in the crystalline state and in aqueous solution have been extensively studied [14-18], as in the following cases where the organisation is due to the guest (Fig. A5 and A6) .


Fig. A5. With guests 1 - 4, organization of cyclodextrins in dimers has been detected in aqueous solutions [14].


Fig A6. Exothermic rotaxanation of Congo red in γCD and endothermic dimerization of the complex [16].

On the other hand, anionic cyclodextrins (in the form of triethyl­ammonium salts) interact with the picrate salt of a positively charged CD to form stable heterodimers in solution (Fig. A7). The association constants, Ka, in DMSO-d6 and in DMSO-d6/H2O (80/20, v/v) range from ~106 M-1 to ~104 M-1. Multivalency in the interactions is manifested by positive cooperativity, negative enthalpy of formation and sizeable negative entropy, in support of development of well ordered supramolecular structures in solution [19].


Figure A7.  Self-assembly of oppositely charged CDs displaying positive cooperativity

III.     Structural and recognition studies on inclusion complexes of cyclodextrins

Complexes with pheromones [20-27] and several plant growth factors [27, 28] have resulted in applications for the protection the olive tree by the controlled release of pheromones of the pests Bactrocera oleae and Prays oleae [29]. The induced fit exhibited by the cavities of permethylated α- and β-cyclodextrins that resulted in the resolution of the racemic mixture of Bactrocera oleae (Scheme A1 and Fig. A8) is very remarkable. [30-31].

                                                       Scheme A1. Spiroacetal (pheromone of Bactrocera oleae)


Fig. A8. (Left): Molecular structure of the complex of permethylated α-CD/(R)-spiroacetal selectively precipitated from the racemic mixture of the guest. (Right): Structure of the complex of permethylated β-CD/(S)-spiroacetal, selectively crystallised from the racemic mixture of the guest. Top: Models of hosts and guests. Bottom: Differential electron density map of the guests and van der Waals map of the hosts.

IV.     Inclusion complexes with drug 

            Several inclusion complexes of drugs in native and derivatised Cds have been studied, such as the NSAID drug acemetacin in βCD [32], penicillin antibiotics (Fig. A9) with natural and carboxylated CDs [33], the antibacterial drug triclosan with native (Fig. A10) and positively and negatively charged CD derivatives [34] and the sulfonylurea hypoglycemic drugs tolbutamide (TBM), tolazamide (TLZ) and glimepiride (GLP), (Fig. A11) into βCD [35].

Fig A9. Ampicillin and the structure of ampicillin in per-6[S-carboxypropionic acid]-γCD (from NMR studies in aqueous solution).

Fig A10. The inclusion complex of the antibacterial agent triclosan in βCD forms dimers packing in channels. The dichlorophenyl ring of the guest enter the cavities of βCD dimers from their primary sides, whereas the hydrophilic chlorophenol ring extends in the space between dimers.

Fig A11. From Left to right the structures of the inclusion complexes of the hypoglycemic drugs TBM, TLZ and GLP in βCD. The mode of inclusion of the drugs is the same: the hydrophobic groups enter the cavities of two hosts, belonging to adjacent βCD dimers from the primary sides, whereas the hydrophilic sulfonylurea moiety is located between dimers, forming H-bonds with the hosts. The packing of the TBM, TLZ complexes is in channel mode, whereas in that of the GLP the βCD dimers are placed further apart (Intermediate mode) due to the increased length of the guest.

V. Inclusion complexes of nucleotides with positively charged cyclodextrins

In contrast to the natural CDs which totally prefer to include the nucleobases of the nucleotides over their ribose rings, the guanidino CDs as well as other positively charged CDs happily encapsulate both ribose and deoxyribose rings in their cavity, whereas the nucleobase moieties are partially included. In several nucleotides studied (Figure A12), we showed that the phosphate groups play an indispensable role in provoking cavity binding through Coulomb interactions with the guanidino groups, as opposed to nucleosides that do not bind at all [36]. Inclusion of nucleotides in the moderately-sized guanidine-β-CD cavity enabled the observation of separate nucleotide conformers, attributed to slow ribose puckering rates. To our knowledge, this has not been reported before


Figure A12. Binding of nucleotides to per(6-guanidino-6-deoxy)­cyclodextrins in aqueous solution


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 B. Synthesis of functional cyclodextrin derivatives and applications

I.  Modified CDs display novel properties

Synthetic modifications of cyclodextrins result in introduction of functional groups that endow the host molecules with specific properties [1-3] ( Fig. B1), allow elongation of the host cavity to capture long guest molecules or the combination of the above that permits direct inclusion in specific direction [4,5] (Fig. B2).

Fig. B1. Synthesis of mono-aminobenzoic acid-substituted β-cyclodextrins and supramolecular self inclusion in the crystalline state [1].


Fig. B2. Per(carboxyethylthio)-β-cyclodextrin encapsulates the elongated dye methyl orange (left) in a sense opposite to that of  natural β-cyclodextrin (right) [5].

II. Positively charged CDs for bio-applications

Per-6-modified CDs with guanidino or lysine- or arginine-like groups (Fig. B3) comprises a very interesting family of hosts that have the ability  to (i) penetrate cell membranes, (ii) transport molecules (iii) compact DNA and therefore perform transfection [6,7]. These properties have been used for transfection of green fluorescent protein (GFP) (Fig. B4) .

Fig. B3. Positively charged CDs

Fig B4. Guanidino and amino cyclodextrins: (left) penetrate cell membranes (fluorescent microscopy image of HeLa cells) and (right) perform DNA transfection that expresses GFP protein (green fluorescence) .

In addition, the above positively charged β- and γ-cyclodextrin derivatives effectively inhibited anthrax toxin action by blocking the transmembrane oligomeric pores formed by the protective antigen (PA63) subunit of the toxin (Fig. B5), whereas α-cyclodextrins were ineffective [8, 9] in collaboration with V. Karginov, Innovative Biologics, USA.


Fig. B5. Positively charged β- and γ-cyclodextrin: A Bacillus anthracis toxin pore blocker

III. Negatively charded CDs with a range of applications

EDTA-type CDs, i.e. CDs modified with aminodiacetyl groups have been synthesized, and characterized (Fig. B6) [10]. Their main property is the ability to coordinate with metal ions, especially lanthanides and particularly gadolinium, Gd(III). The EDTA-CDs coordinate with Gd(III) and fast exchanging water molecules to form metal clusters that display high relaxivity values, especially at high (100 MHz) magnetic fields. Gd-EDTA-CDs exhibit significantly higher relaxivity characteristics compared to existing MRI contrast agents


Fig. B6.  GEDTA (EDTA-γ-cyclodextrin) (center )and its Gd(III) complex bearing four Gd(III) ions (right) as calculated by PM3 semiempirical methods [10]


IV. Glycoclusters

Attachment of recognition sugars in the primary side of cyclodextrins results in formation of glycoclusters (Fig. B7) able to attach on bacterial membranes, thus having possible biomedical applications [11].


Fig. B7. A representative cyclodextrin-based glycocluster with the possibility to recognize cell surface lectins

V. Surface active CDs

A stable, well-packed undecenyl-cyclodextrin (DMBUA) monolayer on Si/SiO2/ novolac resin (AZ) substrate (Fig.  B8) is able to detects triclosan from a 10 nM aqueous solution in a reflectance spectrometer [12].


Fig. B8. Representation of a stable CD monolayer able to detect the antibacterial triclosan (shown schematically).

An elementary supramolecular conducting system was constructed using a novel (±)-thioctic acid-functionalized β-cyclodextrin host deposited on a gold (Au) surface and an iridium-bearing guest molecule with biphenyl tails to insert specifically into the cyclodextrin cavity (Fig. B9). The resulting Au surface functionalised by  this supramolecular system was used to investigate remote electron communication between Au and the Pt/Ir tip of a Scanning Tunneling Microscope. I-V spectroscopic analysis of the tunneling current through the supramolecular layer revealed the relation between the effective height of the barrier and tunneling distance [ 13].


Fig. B9. Representation of the supramolecular system conducting current between the Au substate and the Pt/Ir STM tip. 

VI. Cyclodextrin  derivatives for production of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNOS)

CD derivatives covalently linked with photoactive units provide new systems for molecular transport that combine the inclusion properties of the CDs with photo-triggered properties of the attached unit (multimodal systems). These systems can be employed as photosensitizers (PS) in Photodynamic Therapy (PDT)  for selectively treating neoplastic lesions: PS is accumulated in the tumor and is activated by light of the appropriate wavelength in order to generate reactive oxygen species (ROS), which are highly deleterious to cells.

Most of the PSs used in PDT are porphyrins. The covalent attachment of a CD moiety to a pophyrin can be envisaged as a strategy to combine the drug enacapsulation/solubilization capacity of the CD with the photosensitizing/fluorescence imaging properties of the porphyrin, thus creating a multimodal drug carrier. As an immediate consequence, CD-porphyrin conjugation could enhance the aqueous solubility of the porphyrin and promote its de-aggregation as well as increased fluorescence intensity and lifetime.  The ultimate test of these systems would be their ability to become internalised by cells and preferably display a localisation preference into a certain subcellular compartment.

Thus we have synthesised a porphyrin-βCD conjugate exhibiting the typical red fluorescence of the porphyrin  that encapsulates a nitric oxide photodonor tailor-made to fit the βCD cavity (Fig. B10), which aggregates into a nano-assembly of 16 nm (a supramolecular bichromo­phoric aggregate).  This system retains porphyrin fluorescence thus enabling its imaging into living cells and is able to generate nitric oxide and singlet oxygen under illumination by visible light. It has been proven to be internalized in melanoma cells and induce a significant level of mortality [14], probably due to the combined action of RNOS and ROS , in collaboration with S. Sortino, U of Catania, It.

nano-aggregateChem_Asian J

Fig. B10. A supramolecular nanoaggregate able to produce light-triggered ROS and RNOS

An approach to use protoporphyrin IX (PpIX) for effective application of PDT in cancerous lesions is the topical or systemic administration of 5-aminolevulinic acid (ALA) and its esters, which results in increased production and accumulation of PpIX. The use of  ALA for PpIX biosynthesis is attractive, but has two shortcomings: large concentrations of exogenous ALA (in order to bypass the negative feedback control exerted by heme on enzymatic biosynthesis of PpIX from ALA) and the strong dimerization propensity of ALA.  To circumvent these limitations and possibly enhance the phototoxicity of PpIX by adjuvant chemotherapy, covalent bonding of PpIX with a drug carrier, β-cyclodextrin (βCD) was implemented [15]. The resulting PpIX-βCD has both carboxylic termini of PpIX connected to the CD (Fig. B11). PpIX-βCD is water soluble, that has been found to preferentially localize in mitochondria rather than in lysosomes both in MCF7 and DU145 cell lines, while its phototoxiciy is comparable to that of PpIX. Moreover, PpIX-βCD effectively solubilized the breast cancer drug tamoxifen metabolite N-desmethyltamoxifen (NDTAM) in water. Thus PpIX-βCD is a bimodal βCD derivative and its PpIX-βCD/NDTAM complex has been readily internalized by both cell lines employed (Fig. B11).


Fig. B11. The multimodal action of PpIX-βCD. Phototoxic effect of PpIX and transport of tamoxifen metabolite N-desmethyltamoxifen by βCD.

On another research line, the grafting of SNO groups on the cyclodextrins can afford reactive nitric oxide species (RNOS) generating carriers upon photochemical or thermal stimulation (Fig. B12). SNO-βCDs were characterized in detail for the first time regarding conformational preferences and SNO group content, thermal and photochemical stability, ability to en­capsulate guest molecules as well as cell toxicity and cell permeation [16]. The CD cavity is available for guest encapsulation without noticeable perturbation of the -SNO functionality while hosting e.g. the chemotherapeutic tamoxifen. Nitrosation of per-SH-βCD to form per-SNO-βCD was found to compete with SNO decomposition and disulfide bridge formation, resulting in an average 5.2 SNO groups instead of 7. Mono-SNO-βCD is water soluble, whereas multi-SNO-βCD is DMSO soluble. Both have satisfactory thermal stability, cell permeability and they were found to be chemically non toxic to cells at considerably high incubation concentrations (>200 μM). Thus the combination of RNOS-generating-hosts with RNOS-designed-guest may provide powerful phototoxic systems for PDT applications.


Fig. B12. Left: synthesis of SNO-CDs. Right: bimodal action of a RNOS host carrying a guest molecule 

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C. Structure of macromolecules by X-ray crystallography

X-ray crystallography allows  to “look” at the detailed structure of biological macromolecules in three dimensions at the atomic level. This is essential in order to understand the macromolecules’ function (or malfunction in case of disease), to propose reaction mechanisms or design drugs. Current and past projects include:

 I.   Binding of γ-cyclodextrin to Glucogen Phosphorylase b

Selective binding of γ-cyclodextrin to Glucogen Phosphorylase b (GPb) at the glucogen storage site of the enzyme (Fig. C1) [1]. In order to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, α, β and γ-cyclodextrins (CDs) were identified as moderate mixed-type inhibitors of GPb ( Ki values 47.1, 14.1 and 7.4 mM for α, β and γ-CD, respectively), the γ-CD being better than the others. The structures of GPb with β- and γ-CDs provide an understanding of the binding, which is analogous to this of linear maltopentaose (G5) and maltoheptaose (G7) oligomers namely, anchoring of two glucose residues to GPb. However, the inhibition constant of γ-CD is almost 7-times higher than that of G7 and of β –CD even higher. The lower potency of γ-CD compared to G7 is explained by lack of H-bonds between the γ-CD residues  that are next to the anchoring residues and the protein, due to its round shape.


Fig. C1. γCD (in red) binds to the storage site of the Glucose Phosphorylase b dimer.


II.   Ferredoxins 

Structural and electrochemical studies of the new family of 2[4Fe4S] ferredoxins (Fds) from selected pathogenic bacteria such as  Escherichia coli (EcFd), Pseudomonas aeruginosa (PaFd), Allochromatium vinosum (AlvinFd, Fig. C2) in order to correlate their structure to the widely different reduction potentials of their two metal clusters [–460 (cluster II) and –675 mV (cluster I)] [2,3]. The degree of exposure to the outer environment of cluster I sulfur atoms (Fig. C3) leads towards less negative reduction potentials (E°). This is better illustrated by V13G AlvinFd (high exposure, E° = -594 mV) and EcFd (low exposure, E° = -675 mV). In C57A AlvinFd (Fig. C3a), the movement of the protein backbone, as a result of replacing the non-coordinating Cys57 by Ala, leads to a +50 mV up-shift of the potential of the nearby cluster I, by removal of polar interactions involving the thiolate group and adjustment of the H-bonds network involving the cluster atoms. However, the accurate new structures we have determined for the mutated Alvin Fd, C57A and V13G (at 1.05 and 1.48 Å resolution [3], respectively) indicate the effects of subtle structural differences around cluster II that provide insight on the electrochemical properties. Namely, polar interactions of side chains and water molecules with the sulfur atoms of cluster II  (Fig. C2), which are absent in the environment of cluster I (the cluster with the lowest reduction potential), can be correlated to the ca. 180-250 mV difference between the reduction potentials of clusters I and II.


Fig. C2. The structure of the Alvin Fd variant, C57A (1.05 Å resolution, a). The environment of cluster II, although  buried inside the molecule, is comparable to the exposed cluster II of the widely studied clostridia ferredoxin (b) that has two isopotential clusters (ca. –400 mV) . This is due to a water molecule (W6) entrapped in the interior of the C57A that attracts side chains of polar residues close to the cluster II S-atoms. In contrast, cluster I of C57A is less polar than cluster II of AlvinFd and of clostridia ferredoxins.

Fig. C3. Solvent-accessibility surfaces color-mapped according to the chemical nature of the surface atoms of a, C57A; b, V13G; c, PaFd; d,  EcFd: Nitrogen, oxygen, and carbon atoms are shown in blue, red, and green, respectively. The Cys11SG atoms of cluster I are shown in yellow at the central region of the surfaces. The exposed S1 atom of cluster I of V13G (b) is shown in magenta. The bulky sulfur atom of Met13 of EcFd (d), in orange, limits the exposure of the Cys11SG atom.

III.  Muscle proteins

Collaboration with the group of M. Wilmanns, EMBL-Hamburg on two muscle proteins titin and myomesin

 1.   The structure of the amino terminus titin/telethonin complex [4]: Titin (the largest protein made by human cells) along with myosin and actin, plays a crucial role in the muscle function. The structure of the 2:1 titin:telethonin complex gives the details of the interaction of two titin molecules with telethonin, a small protein-component of the the Z-disc of muscles (Fig. C4). This leads to a model of titin interaction in the Z-disc: two titin molecules from different sarcomeric filaments cross-linked at their amino terminus via telethonin. The study may lead to new insight for some muscle and cardiac diseases; moreover, it may provide a molecular paradigm about the cross-linking of major sarcomeric filaments.

Fig. C4. A: ribbon representation (in two orientations) of the two antiparallel titin immunoglobulin-like domains Z1 (blue, residues 1-98) and Z2 (cyan, residues 99-196 including the Z1-Z2 linker) cross-linked by telethonin (red) via extended antiparallel β-sheets involving the three molecules. B: surface representation of the titin-telethonin-titin complex, in two orientations (in green, the telethonin domain 60-90 not participating in β-sheets). 

  2. The myomesin structure. The mechanical forces exerted by the muscles are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain structures. Myomesin is one such repetitive filament protein, which is thought to form bridges between the main contractile filament, that provides the muscle with resistance in the radial dimension. To investigate how the repetitive structure of myomesin contributes to muscle elasticity, the overall architecture has been determined  using a combination of four complementary structural biology methods [5].  The structure of the myomesin domains my9-my11 has been elucidated by X-ray crystallography. Each domain comprises a immunoglobulin-like and helix pattern (Fig. C5 in green). These have been combined with the already available structure of the final my12-my13 domains of the carboxyl terminus in order to make a model of how the myomesin filament attaches in the M-band of the muscle sarcomer.

Fig. C5. The complete myomesin (My) tail-to-tail filament structure: In ribbon representation the dimeric myomesin IgH domain array My9–My10–My11–My12–(My13)2–My12'–My11'– My10'–My9'. Myomesin domains that have been structurally investigated are shown in violet (first molecule) and blue (second molecule). The helical linkers are shown in green. A ruler provides an overall length estimate of the filament.

IV.    Endoplasmic Reticulum Aminopeptidases

Endoplasmic Reticulum (ER) aminopeptidases ERAP1 and ERAP2 cooperate to trim a vast variety of antigenic peptide precursors to generate mature epitopes for binding onto MHC class I molecules and help regulate the adaptive immune response.  In collaboration with the group of E. Stratikos at our institution, we have determined for the 1st time the structure of ERAP2 to 3.08 Å by X-ray crystallography [6]. The ERAP2 structure (Fig. C6) provides a structural explanation for the different peptide N-terminus specificities between ERAP1 and ERAP2 and suggests that such differences extend throughout the whole peptide sequence. Overall, the structure helps explain how two homologous aminopeptidases cooperate to process a large variety of sequences, a key property to their biological role. Common coding single nucleotide polymorphisms (SNPs) in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. The common ERAP2 SNP rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared to the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. This is primarily due to changes in the catalytic turnover rate (kcat) and not in the affinity for the substrate.  X-ray crystallographic analysis of the ERAP2 392K (Fig. C6)allele suggests that the polymorphism interferes with the stabilization of the N-terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis [7]. The study provides mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in antigen processing enzymes constitutes a component of immune response variability in humans.

Fig. C6. Left: Ribbon representation of ERAP2  colored by domain (domain I in blue, II in green, III in orange and IV in magenta). Right: A, sequence alignment of ERAP2 and homologous aminopeptidases showing conservation of the polymorphic residue 392 (in bold); the adjacent aminopeptidase motif (HELAH) is also indicated; B, key catalytic residues in the ERAP2K structure are shown in stick representation and superimposed with equivalent residues in ERAP2N (2SE6). Note the relative positioning of Lys392 with respect to the catalytic Glu residues as well as the N-terminus of the bound ligand (Lysa). Electron density 2|Fo|-|Fc| at 2σ is indicated around Lys392; C, pairwise electrostatic interaction energies (ΔΕinter in Kcal/mol) calculated between the polymorphic residue 392 and the Lysa ligand. The reported values are the average of the 20 runs with a standard deviation of less than 1%, which represents the uncertainty due to the granularity of the grid; D, superimposition of ERAP2 residues that cap the S1 specificity pocket of the enzyme. |Fo|-|Fc| electron density (at 2.5σ) calculated in the absence of the ligand is shown around Lysa. 2|Fo|-|Fc| electron density at 2σ is indicated around Glu177.

V.    RNA Complexes

Structure determination of complexes of the ribosomal decoding aminoacyl site (A-site) of bacterial 16SrRNA constructs with synthetic analogs of aminoglycosides. Aminoglycoside antibiotics selectively bind to the A-site of bacterial 16SrRNA, interfering in the fidelity of proteosynthesis in the bacterial cells. The designed compounds are synthesised at the collaborating organic Chemical Biology laboratory of our institution headed by Dr D. Vourloumis. Elucidation of the structure of the above compounds in complex with selective RNA constructs that are appropriate models of the A-site, provides the prerequisite for guiding this synthetic effort. Moreover, the structural information combined with biochemical and kinetic experiments is the first step in the development of pharmaceuticals for fighting bacterial infections by RNA-directed therapies.

Fig. C7. Model of the structure of a ligand-free bacterial RNA construct. Three consecutive molecules of RNA monomers (each represented by a different colour) form infinite continuous chains. The structure has been solved by the software IL MILIONE (Burla MC, Caliandro R, Camalli M, Carrozzini B, Cascarano GL, De Caro L, Giacovazzo C, Polidori G, Siliqi, D, Spagna R, J Appl CRYSTALLOG. 2007, 40   609-613)

VI. DsbD: a bacterial thiol-disulfide oxidoreductase

DsbD is one of the five proteins of the bacterial Disulfide bond (Dsb) formation system. It is located in the inner membrane of Gram-negative bacteria and it is responsible for transporting reducing power from the cytoplasm to the oxidising periplasm of the cell (Fig. C8). It comprises three domains; the central transmembrane domain (tmDsbD), of unknown structure, is flanked by two globular periplasmic domains, the N-terminal domain nDsbD) with an immunoglobulin-like fold and the C-terminal domain (cDsbD) with a classical thioredoxin (Trx) fold. DsbD plays an important role in oxidative protein folding because it allows for the correct formation of disulfide bonds in proteins functioning in harsh extracytoplasmic environments [8]. It is also involved in bacterial pathogenesis as the expression and stability of most virulent factors (secreted molecules, secretion apparatuses, adhesion systems etc) are dependent on the presence of DsbD. The structures of the N-terminal domain in the reduced form and of two point-mutants of the C-terminal domain have been determined [9,10] in collaboration with the Department of Biochemistry, University of Oxford (Prof. Stuart J. Ferguson, Prof. Christina Redfield and Dr Despoina A.I. Mavridou). This work contributed significantly in elucidating the interaction of the soluble domains of this unique oxidoreductase but also in the general understanding of the factors controlling the reactivity of the ubiquitous thioredoxin fold.


Fig. C8. DsbD: The sole reductant provider in the periplasm.


VI. Macromolecular crystallization methods

New crystallisation methodology for biological macromolecules, in collaboration with Imperial College London.

1. As Coordinators of the Industry-Academia Partnerships and Pathways - Marie Curie Project TOPCRYST, we developed the use of Dual Polarization Interferometry (Fig. C9), pioneered by Farfield Scientific Ltd., to probe crystallisation at its most crucial stages. This allows to predict the outcome of crystallisation trials when they are still at their earliest stages and thus to rationally design and direct such experiments in order to lead them to well-diffracting crystals [11]. Research on other techniques for effective a priori prediction of crystallisation conditions, such as the use of Genetic Algorithms and of calorimetry, is also being actively pursued [12].


Fig. C9. Optical principle of dual polarization interferometry. Light from a sensing waveguide in contact with the investigated solution interferes with that from a reference waveguide, resulting in interference fringes characterized by their phase and contrast.


2. Research into substances and materials promoting the heterogeneous nucleation of macromolecular crystals is a blossoming topic in crystallogenesis. We have participated in developing the use of materials containing pores of cavities, such as Bioglass, carbon nanotubes, and Molecularly Imprinted Polymers (Fig. C10), as heterogeneous nucleants and are pursuing further ideas [13-16].

crystals from nucleants

Fig. C10. Crystals of a protein (human macrophage Migration Inhibiting Factor) growing at metastable conditions in the presence of Molecularly Imprinted Polymer imprinted with protein


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