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Glycosaminoglycans (GAGs) are linear, highly sulfated polysaccharides expressed by almost all animal cells. They occur as soluble molecules, or form proteoglycans by being O-linked to different core proteins on the cell surface and in the extracellular matrix. Due to their ability to interact with diverse proteins and to modulate their biologic functions, GAGs are main drivers of mammalian biology. However, to the present day, the human GAG binding proteome has only been insufficiently explored. The aim of this study was therefore to investigate the human GAG binding proteome of different sources by using the major GAG classes as ligands, and to explore the GAG-binding selectivity of the human plasma proteome. For this purpose, proteins were pulled down from immobilized low molecular weight heparin, heparan sulfate, and dermatan sulfate under different conditions and were identified by nano-LC/MS². Four hundred and fifty eight human GAG binding proteins have been identified, whereas plasma proteins showed clear differences in their GAG-binding specificity/selectivity and affinity. We were able to differentiate between proteins that bound to all three glycan ligands and proteins that showed selective binding to one or two glycan ligands. Moreover, step-gradient salt elution revealed different binding affinities toward different GAG ligands. On top of proteins with well-known GAG-binding properties we have identified formerly unknown GAG binders. Functional annotation of the identified GAG-binding proteins showed clusters of proteins that are involved in a variety of biological processes. The method described here is well suited for identifying GAG-binding proteins and for comparing human subproteomes with respect to binding to different GAG classes.

The ELR(-)CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74-103)) being the most potent. The COOH-terminal peptide CXCL9(74-103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74-103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74-103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74-103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions. ispartof: Journal of Biological Chemistry vol:290 issue:35 pages:21292-304 ispartof: location:United States status: published

We have engineered GPCR (G protein-coupled receptor) knock-out and high GAG-binding affinity into CXCL12α to inhibit CXCL12α-induced cell migration. Compared to wtCXCL12, the mutant CXCL12α (Δ8 L29K V39K) exhibited a 5.6-fold and a 2.2-fold affinity increase for heparin and heparan sulfate, respectively. From NaCl-based heparin displacement chromatography we concluded that more amino acid replacements would lead to altered GAG (glycosaminoglycan) ligand specificity. GAG silencing by this mutant was shown in a murine seeding model of human cancer cells, whereby a greatly reduced number of liver metastases was detected when the animals were treated intravenously with 1mg/kg CXCL12α (Δ8 L29K V39K) before cancer cell application.

The biological activity of chemokines requires interactions with cell surface proteoglycans. We have determined the structure of the chemokine RANTES (regulated on activation normal T cell expressed) in the presence of heparin-derived disaccharide analogs by X-ray crystallography. These structures confirm the essential role of the BBXB motif in the interaction between the chemokine and the disaccharide. Unexpected interactions were observed in the 30s loop and at the amino terminus. Mutant RANTES molecules were designed to abrogate these interactions and their biological activity examined in vivo. The K45E mutant within the BBXB motif lost the capacity to bind heparin and the ability to elicit cellular recruitment. The Y3A mutant maintained its capacity to bind heparin but was unable to elicit cellular recruitment. Finally, a tetrasaccharide is the smallest oligosaccharide which effectively abolishes the ability of RANTES to recruit cells in vivo. These crystallographic structures provide a description of the molecular interaction of a chemokine with glycosaminoglycans.

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.

Binding of interleukin-8 (IL-8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running approximately 15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL-8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL-8 single-site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL-8 > IL-8(H18A) >> IL-8(R68A) > IL-8(K67A) >> IL-8(K20A) > IL-8(R60A) >> IL-8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL-8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL-8 monomers in the dimeric chemokine by an N-acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N-terminal alpha-helices of IL-8 like a horseshoe. A 5-ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL-8 with high affinity.

Application of reverse transcription-PCR to total RNA prepared from TNF-alpha (tumour necrosis factor-alpha)-stimulated HUVECs (human umbilical vein endothelial cells) revealed that the syndecan-2 mRNA was up-regulated by this inflammatory stimulus. By immunoprecipitation using an anti-syndecan-2 antibody on TNF-alpha-stimulated HUVEC lysates, inflammation-induced interleukin-8 was found to be an interaction partner of this HS (heparan sulphate) proteoglycan, but not of any other syndecan on these cells. The glycosylated [Syn2(ect)(+HS)] and non-glycosylated [Syn2(ect)(-HS)] forms of Syn2(ect) (the syndecan-2 ectodomain) were purified from a stably transfected human cell line and from a bacterial expression system respectively. By CD spectroscopy, Syn2(ect) was found to adopt an all-beta secondary structure. The dissociation constant of Syn2(ect)(+HS) with respect to interleukin-8 binding was determined by isothermal fluorescence titrations to be 23 nM. Despite its lack of HS chains, Syn2(ect)(-HS) exhibited significant binding to the chemokine, with a K (d) of >1 microM. Thus, in addition to glycosaminoglycan binding, protein-protein contacts might also contribute to the chemokine-proteoglycan interaction.

Tandem mass spectrometry coupled to HPLC is the state of the art technique in proteomic research. Here we describe a highly sensitive nano liquid chromatography system (nano HPLC) for analysis of protein digests. Using preconcentration in a column-switching set-up, we were able to inject large sample volumes (or =250 microL) without significant loss of sensitivity. The major problem with this type of preconcentration is usually the occurrence of void volumes. In order to diagnose void volumes a simple and easy test was developed by which the UV trace and the pressure profile in the separation column were monitored. Part by part replacement of connection tubing restored a void volume-free system. A major pre-requisite for handling samples in the femtomol range was found to be the use of protein/peptide-saturated columns tryptic digests of cytochrome C were injected directly onto the reversed-phase nano separation column (75 microm inner diameter) and the separation results were compared with chromatograms obtained from separations using column switching. By using column switching we were able to inject large sample volumes in a short time period without losing resolution.

1-Unsubstituted 4-dimethylamino-5,6-dihydropyridine-2(1 H )-thiones were converted to isomeric piperidin-4-ols which were separated and N -methylated to 2-substituted 1-methylpiperidin-4-ols. Their 1-phenyl analogues were prepared from 4-dimethylamino-5,6-dihydro-1-phenylpyridine-2(1 H )-thiones. After their conversion to dihydro-1-phenylpyridin-4(1 H )-ones the hydrogenation gave isomeric 1-phenylpiperidin-4-ols which were separated. O -Alkylation of the 1-substituted piperidin-4-ols by various methods yielded 2-substituted analogues of diphenylpyraline. Their antimycobacterial activity was examined. The configurations and conformations of the piperidine derivatives were investigated by NMR spectroscopy.

The cloning, purification, and biophysical characterization of the first eukaryotic cold shock protein homologue, Cla h 8, expressed as single functional polypeptide is reported here. It was discovered as a minor allergen of the mold Cladosporium herbarum by phage display using a library selectively enriched for IgE-binding proteins. Based on the sequence homology of Cla h 8 with bacterial cold shock proteins (CSPs), a homology-based computer model of the allergen was computed indicating an all-beta structure of Cla h 8. This major structural feature was confirmed by CD spectroscopy. Despite the structural similarities with bacterial CSPs, the DNA-binding and unfolding behavior of Cla h 8 exhibited unique and previously undescribed characteristics. High affinities of Cla h 8 for single-stranded DNA as well as for double-stranded DNA corresponding to the human Y-box were detected. The affinity for double-stranded DNA increased significantly with decreasing temperature, which was paralleled by an increase in the beta sheet content of the protein. Temperature-dependent fluorescence anisotropy and far-UV CD measurements revealed different unfolding transitions at 28 and at 35.7 degrees C, respectively, indicating a multistate transition, which is uncommon for CSPs. The enhanced affinity for DNA at low temperatures together with the low unfolding transition refer to the functional significance of Cla h 8 at reduced temperatures.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS), but its function is not fully understood. Specifically, it is unclear whether BH4 participates directly in electron transfer. We investigated the redox properties of BH4 and several other pteridines with cyclic voltammetry and Osteryoung square wave voltammetry. BH4 was oxidized at a potential of +0.27 V vs normal hydrogen electrode (NHE); the corresponding reductive signal after the reversal of the scan direction was very small. Instead, reduction occurred at a potential of −0.16 V vs NHE; there was no corresponding oxidative signal. These two transitions were interdependent, indicating that the reductive wave at −0.16 V represented the regeneration of BH4 from its product of oxidation at +0.27 V. Similar voltammograms were obtained with tetrahydroneopterin and 6,7-dimethyltetrahydropterin, both of which can substitute for BH4 in NOS catalysis. Completely different voltammograms were obtained with 7,8-dihydrobiopterin, sepiapterin, 2′-deoxysepiapterin, and autoxidized BH4. These 7,8-dihydropterins, which do not sustain NOS catalysis, were oxidized at much higher potentials (+0.82–1.04 V vs NHE), and appreciable reduction did not occur between +1.2 and −0.8 V, in line with the concept of a redox role for BH4 in NOS catalysis. However, the electrochemical properties of the potent pterin-site NOS inhibitor 4-amino-BH4 resembled those of BH4, whereas the active pterin cofactor 5-methyl-BH4 was not re-reduced after oxidation. We conclude that the 2-electron redox cycling of the pterin cofactor between BH4 and quinonoid dihydrobiopterin is not essential for NO synthesis. The data are consistent with 1-electron redox cycling between BH4 and the trihydrobiopterin radical BH3•.

The fluorescence intensity of the two flavin prosthetic groups, FMN and FAD, in neuronal nitric oxide synthase (nNOS) was found to decay highly nonexponentially, being best described by four fluorescence lifetimes. This excited state heterogeneity is the result of multiple flavin quenching sites which are due to several flavin microenvironments created mainly by stacking with aromatic amino acids. Investigating nNOS in the absence of one or more of Ca2+/calmodulin, tetrahydrobiopterin, and heme revealed an influence of these cofactors on the microenvironments of the flavin prosthetic groups. Similar effects on the flavin rotational dynamics were found by analyzing the fluorescence anisotropy decay of the holo and of the different apo forms of nNOS. Since the tetrahydrobiopterin and the heme are located in the N-terminal oxygenase domain of nNOS, their effect on the flavins in the C-terminal reductase domain is explained by a folding back of the reductase domain onto the oxygenase domain. Thereby a domain-domain interface is created containing the FAD, FMN, heme, and tetrahydrobiopterin prosthetic groups which allows for efficient electron transfer during catalysis. The heme group, which is known to be essential for homodimerization of nNOS, was also found to be essential for the formation of the domain-domain interface.

Specific immunotherapy is an efficient treatment for patients suffering from type I allergy. The mechanisms underlying successful immunotherapy are assumed to operate at the level of T helper cells, leading to a modulation of the immune response to allergens. During immunotherapy, increasing doses of allergens are given on a regular basis, and the beneficial effects for the patient depend on the concentration of allergen used. On the other hand, the risk of IgE-mediated anaphylactic side effects also increase with the amount of allergen applied per injection. Therefore, we have proposed the use of hypoallergenic (low IgE binding activity) forms of allergens for immunotherapy. We evaluated by site-directed mutagenesis the contributions of individual amino acid residues/positions for IgE binding to Bet v 1, the major allergen of birch pollen. We found that IgE binding to Bet v 1 depended on at least six amino acid residues/positions. Immunoblot analyses and inhibition experiments showed that the multiple-point Bet v 1 mutant exhibited extremely low reactivity with serum IgE from birch pollen-allergic patients. In vivo (skin prick) tests showed that the potency of the multiple-point mutant to induce typical urticarial type I reactions in pollen-allergic patients was significantly lower than for wild-type Bet v 1. Proliferation assays of allergen-specific T cell clones demonstrated that these six amino acid exchanges in the Bet v 1 sequence did not influence T cell recognition. Thus, the Bet v 1 six-point mutant displayed significantly reduced IgE binding activity, but conserved T cell activating capacity, which is necessary for immunomodulation. The approach described here may be generally applied to produce allergen variants to be used in a safe therapy form of immediate-type allergies.

The interaction of chemokines and GAGs (glycosaminoglycans) on endothelial surfaces is a crucial step for establishing a chemotactic gradient which leads to the functional presentation of chemokines to their GPCRs (G-protein-coupled receptors) and thus to activation of approaching leucocytes. Based on molecular modelling, biophysical investigations, cell-based and in vivo experiments, we have developed a novel concept for therapeutically interfering with chemokine–GAG interactions, namely dominant-negative chemokine mutants with improved GAG binding affinity and knocked-out GPCR activity. These recombinant proteins displace their wild-type chemokine counterparts from the natural proteoglycan co-receptors without being able to activate leucocytes via GPCRs. Our mutant chemokines therefore represent the first protein-based GAG antagonists with high therapeutic potential in inflammatory diseases. Abbreviations: GAG, glycosaminoglycan; GPCR, G-protein-coupled receptor; IL-8, interleukin-8

IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.

IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.

Non-exponential fluorescence decays due to the photochemical reaction of 10-cyano-9-tert-butyl-anthracene towards the Dewar isomer are analyzed to gain information on the free volume of a polymer glass (poly- n -butyl-methacrylate) at different temperatures. The simple model of a multiexponential fitting procedure is well suited to following the temperature dependence in the envelope of this distribution qualitatively, but it fails to detect finer structures. More demanding methods (maximum entropy and exponential series) result in at least trimodal distributions indicating preferred polymer free volume voids within a broad distribution. Increasing temperature favours the decay time range associated with the largest free volume void.

Nurnberger S, Miller I, Duvigneau JC, Kavanagh ET, Gupta S, Hartl RT, Hori O, Gesslbauer B, Samali A, Kungl A, Redl H, Kozlov AV. Impairment of endoplasmic reticulum in liver as an early consequence of the systemic inflammatory response in rats. Am J Physiol Gastrointest Liver Physiol 303: G1373-G1383, 2012. First published October 11, 2012; doi: 10.1152/ajpgi.00056.2012.-It is well known that systemic inflammatory response (SIR) often causes liver dysfunction. The aim of this study was to identify the intracellular compartment in the liver most susceptible to SIR. We analyzed morphology, ultrastructure, proteome, and expression of relevant genes in livers of rats subjected to endotoxic shock. Histological examination revealed that focal necrosis in liver was insignificant to explain liver dysfunction. Electron microscopy revealed no morphological changes in the mitochondrial structure and in the cytosol, but dilated endoplasmic reticulum (ER) cisterns were frequently observed. Apoptosis was found in white blood cells within liver tissue but not in hepatocytes. Mitochondrial, ER, and cytosolic fractions were subjected to proteome analysis by difference gel electrophoresis, and the protein spots with the highest degree of differential regulation were identified with mass spectrometry. The most pronounced proteome changes appeared in the ER, manifested as a remarkable downregulation of several proteins essential for ER functions, such as protein synthesis and transport, whereas the changes in mitochondrial and cytosolic fractions suggested a compensatory response. ER stress, as an underlying mechanism for ER impairment, was confirmed by analysis of upstream (splicing X-box-binding protein 1 mRNA) and downstream (e.g., 78-kDa glucose-regulated protein mRNA) markers, suggesting ongoing unresolved ER stress as a cause for ER dilation. Because ER is the intracellular compartment responsible for the major liver functions, our data suggest that inflammatory mediators induce unresolved ER stress, resulting in the biochemical, functional, and morphological impairment of ER that in turn causes liver dysfunction. The pathway activating ER stress in response to SIR is not known yet.

Glycosaminoglycans (GAGs) are a class of highly negatively charged, unbranched, O-linked polysaccharides that are involved in many diseases. Their role as a protein-binding matrix on cell surfaces has long been recognized, but therapeutic approaches to interfere with protein-GAG interactions have been limited due to the complex chemistry of GAGs, on one hand, and due to the lack of specific antibodies against GAGs, on the other hand. We have developed a protein engineering platform (the so-called CellJammer(®) technology), which enables us to introduce higher GAG-binding affinity into wild-type GAG-binding proteins and to combine this with impaired biological, receptor-binding function. Chemokines are among the prototypic GAG-binding proteins and here we present selected results of our CellJammer technology applied to several of these proinflammatory proteins. An overview is given of our lead decoy protein, PA401, which is a CXCL8-based mutant protein with increased GAG-binding affinity and decreased CXCR1/2 binding and activation. Major results from our CCL2 and CCL5 programmes are also summarized and the potential for clinical application of these decoy proteins is presented.

The versatility of the surface of Borrelia, the causative agent of Lyme borreliosis, is very important in host-pathogen interactions allowing bacteria to survive in ticks and to persist in a mammalian environment. To identify the surface proteome of Borrelia, we have performed a large comparative proteomic analysis on the three most important pathogenic Borrelia species, namely B. burgdorferi (strain B31), B. afzelii (strain K78), and B. garinii (strain PBi). Isolation of membrane proteins was performed by using three different approaches: (i) a detergent-based fractionation of outer membrane proteins; (ii) a trypsin-based partial shedding of outer cell surface proteins; (iii) biotinylation of membrane proteins and preparation of the biotin-labelled fraction using streptavidin. Proteins derived from the detergent-based fractionation were further sub-fractionated by heparin affinity chromatography since heparin-like molecules play an important role for microbial entry into human cells. All isolated proteins were analysed using either a gel-based liquid chromatography (LC)-MS/MS technique or by two-dimensional (2D)-LC-MS/MS resulting in the identification of 286 unique proteins. Ninety seven of these were found in all three Borrelia species, representing potential targets for a broad coverage vaccine for the prevention of Lyme borreliosis caused by the different Borrelia species.

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.

The potent activator and chemoattractant for human neutrophils, neutrophil-activating peptide 2 (NAP-2), has been cloned and expressed in Escherichia coli. The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC. The biological activity of recombinant human NAP-2 (rhNAP-2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP-2. rhNAP-2 has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P222 with unit cell dimensions of a = 30.8 Å, b = 39.5 Å and c = 95.3 Å. A packing density of 3.8 Å3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed. The crystals were shown to diffract to beyond 2.0 Å on a conventional X-ray source. They are stable to X-rays for several days and are thus suitable for high resolution structure determination.

Summary The inherent cytotoxicity of aberrantly folded protein aggregates contributes substantially to the pathogenesis of amyloid diseases. It was recently shown that a class of evolutionary conserved proteins, called MOAG-4/SERF, profoundly alter amyloid toxicity via an autonomous but yet unexplained mode. We show that the biological function of human SERF1a originates from its atypical ability to specifically distinguish between amyloid and nonamyloid aggregation. This inherently unstructured protein directly affected the aggregation kinetics of a broad range of amyloidogenic proteins in vitro, while being inactive against nonamyloid aggregation. A representative biophysical analysis of the SERF1a:α-synuclein (aSyn) complex revealed that the amyloid-promoting activity resulted from an early and transient interaction, which was sufficient to provoke a massive increase of soluble aSyn amyloid nucleation templates. Therefore, the autonomous amyloid-modifying activity of SERF1a observed in living organisms relies on a direct and dedicated manipulation of the early stages in the amyloid aggregation pathway., Graphical Abstract Highlights ► SERF1a drives the assembly of amyloidogenic proteins ► SERF1a discriminates between amyloid and nonamyloid aggregation ► SERF1a acts through an early interaction with α-synuclein amyloid precursors ► SERF1a catalyzes the formation of transient α-synuclein “on-pathway” aggregates, The protein class called MOAG-4/SERF profoundly alters amyloid protein toxicity via an autonomous and previously unidentified pathway. Falsone and colleagues demonstrate that the amyloid-modifying ability of human SERF1a is driven by a transient interaction with early precursors in the amyloid pathway. As a consequence, SERF1a promotes amyloid aggregation of several amyloidogenic proteins, while being insensitive to unspecific protein aggregation. This identifies SERF1as a specialized amyloid factor, in support to its autonomous mode of action observed in cells.

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, (57)KSKQAR(62)) along with Lys(17), significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.

We have investigated the time-resolved fluorescence properties ofll-dityrosine in aqueous solution. Typically, three exponential components were needed to fit the fluorescence pattern adequately, with pure decay terms for the low-intensity, high-energy state (λem = nm) but with a pronounced subnanosecond rise phase for the predominant red-edge fluorescence (λem > 380). Dual fluorescence behavior is indicative of an intramolecular precursorsuccessor pair, i.e., a consecutive intramolecular excited-state reaction. We suggest that this reaction is a torsional motion of the (deprotonated) monoanion along the biphenolic bond. Analysis of the fluorescence anisotropy decay of dityrosine yielded two rotational correlation times, the longer of which is associated with a negative preexponential term. The increase with time in the horizontally polarized component of the intensity decay is interpreted as the result of an electronic rearrangement in the excited state when the successor form of dityrosine is generated. Lifetime distributions of experimental data were probed by an unbiased exponential series method which uses a Tikhonov-type regularization function. The procedure revealed three well-separated groups of lifetimes, the short-lived ensemble forming a formally negative distribution. A photophysical model is introduced which interprets the biexponential decay of dityrosine in terms of overlapping emission signals from the precursor and the successor molecule.

Chemokine function in vivo depends on the presentation by structures of the extracellular matrix or on endothelial surfaces. CCL5 contains two clusters of basic amino acid residues ( 44 RKNR 47 and 55 KKWVR 59 ) implicated in presentation of the protein. While 44 RKNR 47 has been shown to moderate CCL5 binding to glycosaminoglycans (GAGs), no direct role for the basic residues in the so called 50s loop ( 55 KKWVR 59 ) as a presentation structure has been published to date. In ex vivo studies both regions were found to be necessary for direct tissue binding suggesting a role for 55 KKWVR 59 . In vitro T lymphocyte and monocyte induced firm adhesion under flow, as well as leukocyte recruitment to the peritoneal cavity in vivo was reduced in the 50s mutant. The binding of the 50s mutant to endothelial cells was significantly reduced as compared to the wild type protein demonstrated by ELISA. The 50s mutant had little impact on GAG binding in vitro . These data suggest that functional CCL5 presentation is mediated through both the 40s as well as the 50s loop with differential functions of the two loops of clusters of basic residues.

Acute renal allograft damage is caused by early leukocyte infiltration which is mediated in part by chemokines presented by glycosaminoglycan (GAG) structures on endothelial surfaces. CXCL8 can recruit neutrophils and induce the firm arrest of monocytes on activated endothelial cells. A human CXCL8-based antagonist (dnCXCL8) designed to generate a dominant-negative mutant protein with enhanced binding to GAG structures and reduced CXCR1/2 receptor binding ability was tested in models of early allograft injury. The agent displayed enhanced binding to GAG structures in vitro and could antagonize CXCL8-induced firm adhesion of monocytes as well as neutrophils to activated microvascular endothelium in physiologic flow assays. In a rat model of acute renal damage, dnCXCL8 treatment limited proximal tubular damage and reduced granulocyte infiltration. In a Fischer 344 (RT1(lvl)) to Lewis (RT1(l)) rat acute renal allograft model, dnCXCL8 was found to reduce monocyte and CD8+ T-cell infiltration into glomeruli and to limit tubular interstitial inflammation and tubulitis in vivo. Early treatment of allografts with agents like dnCXCL8 may help reduce acute allograft damage and preserve renal morphology and thereby help limit chronic dysfunction.

Binding to glycosaminoglycans (GAGs) is a necessary prerequisite for the biological activity of the proinflammatory chemokine RANTES in vivo. We have applied protein engineering methods to modulate equilibrium-binding affinity as well as binding kinetics of RANTES towards its GAG ligand which also altered the chemokine's oligomerization behavior. Out of 10 mutants, A22K and H23K were chosen for further in vitro and in vivo characterization because their stability was comparable with wild-type (wt) RANTES. In chemical cross-linking experiments, A22K gave higher and H23K lower molecular weight aggregates compared with wtRANTES as shown on SDS-PAGE. All mutants contained an N-terminal methionine residue, a well-described G-protein-coupled receptor (GPCR) antagonistic modification, which resulted in the mutants' inability to induce monocyte chemotaxis. In surface plasmon resonance experiments using immobilized heparan sulfate (HS) and physiological buffer conditions, Met-RANTES exhibited a significantly longer residual time on the GAG chip compared with the other RANTES variants. In Scatchard plot analysis, RANTES gave a bi-phasic, bell-shaped curve suggesting 'creation' of ligand-binding sites on the protein during HS interaction. This was not observed in the mutants' Scatchard plots which gave K(d) values of 317.5 and 44.5 nM for the A22K and H23K mutants, respectively. The mutants were subsequently tested for their inhibitory effect in a rat model of autoimmune uveitis where only H23K exhibited a transient improvement of the clinical disease score. H23K is therefore proposed to be a GPCR-inactive GAG antagonist which displaces the wt chemokine from its natural HS-proteoglycan co-receptor. The protein engineering approach presented here opens new ways for the treatment of RANTES-related diseases.

Glycosylation is the most common form of post-translational modifications by which oligosaccharide side chains are covalently attached to specific residues of the core protein. Especially O-linked glycan structures like the glycosaminoglycans were found to contribute significantly to many (patho-)biological processes like inflammation, coagulation, cancer and viral infections. Glycans exert their function by interacting with proteins thereby changing the structure of the interacting proteins and consequently modulating their function. Given the complex nature of cell-surface and extracellular matrix glycan structures, this therapeutic site has been neglected for a long time, the only exception being the antithrombin III-glycan interaction which has been successfully targeted by unfractionated and low-molecular weight heparins for many decades. Due to the recent breakthrough in the '-ome' sciences, among them proteomics and glycomics, protein-glycan interactions became more amenable for therapeutic approaches so that novel inhibitors of this interaction are currently in preclinical and clinical studies. An overview of current approaches, their advantages and disadvantages, is given and the promising potential of pharmacologically interfering with protein-glycan interactions is highlighted here.

Atherosclerosis is characterized by chronic inflammation of the arterial wall due to chemokine-driven mononuclear cell recruitment. Activated platelets can synergize with chemokines to exacerbate atherogenesis; for example, by deposition of the chemokines platelet factor-4 (PF4, also known as CXCL4) and RANTES (CCL5), triggering monocyte arrest on inflamed endothelium. Homo-oligomerization is required for the recruitment functions of CCL5, and chemokine heteromerization has more recently emerged as an additional regulatory mechanism, as evidenced by a mutual modulation of CXCL8 and CXCL4 activities and by enhanced monocyte arrest resulting from CCL5-CXCL4 interactions. The CCL5 antagonist Met-RANTES reduces diet-induced atherosclerosis; however, CCL5 antagonism may not be therapeutically feasible, as suggested by studies using Ccl5-deficient mice which imply that direct CCL5 blockade would severely compromise systemic immune responses, delay macrophage-mediated viral clearance and impair normal T cell functions. Here we determined structural features of CCL5-CXCL4 heteromers and designed stable peptide inhibitors that specifically disrupt proinflammatory CCL5-CXCL4 interactions, thereby attenuating monocyte recruitment and reducing atherosclerosis without the aforementioned side effects. These results establish the in vivo relevance of chemokine heteromers and show the potential of targeting heteromer formation to achieve therapeutic effects.

2-Substituted derivatives of the antihistaminic agents Bamipine, Diphenylpyraline and of their 1-phenyl analogues were tested for their antimycobacterial and H1-antagonistic activities. They are strong H1-receptor antagonists and also inhibit the growth of mycobacterials with a maximum MIC of 6.25 μg/mL against Mycobacterium tuberculosis H37Rv. H1-receptor antagonistic potency was slightly decreased by substitution in ring position 2 and distinctly diminished by N-aryl substitution. The antimycobacterial potency of Diphenylpyraline was in general increased by substitution in ring position 2, whereas only a few Bamipine derivatives showed markedly improved activity. A correlation between the two activities was not detected for those compounds.

Defining protein-protein interaction networks is a major goal of proteomics. Here, we present a protocol for coimmunoprecipitation, a technique suitable for the isolation of whole protein complexes in vivo and their subsequent identification by either immunoblotting or mass spectrometric sequencing combined to database search.

Defining protein-protein interaction networks is a major goal of proteomics. Here, we present a protocol for coimmunoprecipitation, a technique suitable for the isolation of whole protein complexes in vivo and their subsequent identification by either immunoblotting or mass spectrometric sequencing combined to database search.

Glycosylation is the most frequent PTM and contributes significantly to the function of proteins depending on the type of glycosylation. Especially glycan structures like the glycosaminoglycans are considered to constitute themselves the major function of the glycoconjugate which is therefore termed proteoglycan. Here we review recent views on and novel tools for analysing the proteoglycanome, which are directly related to the type of glycanation under investigation. We define the major function of the proteoglycanome to be its interaction with various proteins in many different (patho-)physiological conditions. This is exemplified by the differential glycosaminoglycan-interactome of healthy versus arthritic patient sera.

3-O-sulfation of heparan sulfate (HS) is the rarest modification within heparan sulfate biosynthesis resulting in unique biological activities. Heparan sulfate d-glucosaminyl 3-O-sulfotransferase-3A (3-OST-3A) (EC 2.8.2.23) generates a binding site for the envelope glycoprotein D (gD) of herpes simplex virus 1. We have expressed the sulfotransferase domain of the human heparan sulfate 3-OST-3A isoform in Escherichia coli and subsequently purified the active enzyme which was found to be present as an oligomer under nonreducing conditions. The activity of the enzyme was tested by a novel gD-dependent gel mobility assay. A biophysical characterisation of 3-OST-3A was performed to study ligand binding and ligand-induced structural changes. Interestingly, the natural substrate HS did not cause a secondary structural change in the enzyme, whereas heparin and chondroitin sulfate did, both of which also exhibited similar high affinity binding to 3-OST-3A compared to HS as detected by isothermal fluorescence titrations. In cross-link assays, only HS was found to induce high molecular aggregates of 3-OST-3A whereas other GAG ligands did not or even inhibited enzyme oligomerisation like the K5 polysaccharide, which was nevertheless found to bind to the enzyme. We therefore conclude that since 3-OST-3A is able to bind also non-substrate GAG ligands with high affinity, discrimination among ligands is triggered by protein oligomerisation.

The science of decoding the information contained within complex glycan structures is commonly referred to as 'glycomics'. Information flow is usually governed by the binding of proteins to these glycan structures, resulting in the induction of biological processes such as cell migration and activation. Therapeutic approaches which interfere with this glyco-information flow are described.

VEGF165 binding to endothelial cells is potentiated by glycosaminoglycans (GAGs). Here, we have investigated the impact of VEGF165 N-glycosylation on GAG binding. Although glycosylated VEGF165 bound to heparin with only slightly higher affinity than non-glycosylated VEGF165, the natural ligand heparan sulfate induced a conformational change only in the glycosylated protein. Unfolding studies of the VEGF proteins indicated a stabilising effect of heparin on the growth factor structure.

For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.

CD34+ preparations from five different umbilical cord samples were compared with respect to their proteome profile using 2-D gel electrophoresis. Fifty-two protein spots were found to match in all preparations referring to the high heterogeneity of such samples indicating a not fully developed (or instable) proteome of stem cells. All matching spots were subjected to in-gel digestion and nano-LC-MS/MS sequence analysis, from which 22 proteins were unambiguously identified.

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.

Binding of interleukin-8 (IL-8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running approximately 15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL-8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL-8 single-site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL-8IL-8(H18A)IL-8(R68A)IL-8(K67A)IL-8(K20A)IL-8(R60A)IL-8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL-8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL-8 monomers in the dimeric chemokine by an N-acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N-terminal alpha-helices of IL-8 like a horseshoe. A 5-ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL-8 with high affinity.

We investigated in vivo effects of norepinephrine (NE) on the transcription of 200 immunologically relevant genes in the mouse. Balb/c mice were s.c. implanted with NE containing retard tablets. Twelve hours later, splenic mRNA was prepared and hybridized onto cDNA microarrays containing the sequences of the major cytokines, their receptors and all CD-antigens of the mouse. Consistent results were obtained with a set of five genes: in the NE-treated animals four genes (CXCR4, VCAM1, IL-1R2, CD 14) were found 2-8 fold upregulated as compared to sham treated animals, whereas the gene for CCR3 was downregulated (0.5 fold). The findings were confirmed using quantitative reverse transcriptase Real Time PCR. These first results prove the usefulness of gene microarray technology towards transcription pattern analysis in neuroimmune interactions. Furthermore, they support the relevance of catecholamines in the regulation of leukocyte migration and the inflammatory response.

Stem cells represent a promising tool for the treatment of various hematopoietic diseases. In order to identify stem cell-specific proteins, the proteome of human stem cells from umbilical cord blood was explored for the first time. For this purpose, the crude lysate of 4 × 105 CD34+ cells was subjected to in solution trypsin digestion. The resulting peptides were then separated via cation exchange followed by reversed phase chromatography and analyzed by nanospray MS/MS. Database search revealed a total of 215 proteins which could be reliably identified. To obtain a more complete picture of the human stell cell proteome and to also access low abundant proteins, pooling of more than one CD34+ preparations seems necessary in order to increase the cell number and thus the protein content.

The binding of interleukin-8 (IL-8) to heparan sulfate (HS) proteoglycans on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Fluorescence anisotropy measurements yielded an IL-8 dimerization constant of 120 nM. The binding affinities, obtained by isothermal fluorescence titration, of size-defined heparin and HS oligosaccharides to the chemokine were found to depend on the oligomerization state of IL-8: high affinity was detected for monomeric and low affinity was detected for dimeric IL-8, referring to a self-regulatory mechanism for its chemoattractant effect. The highest affinity for monomeric IL-8 was detected for the HS octamer with a K(d)5 nM whereas the dissociation constants of dimeric IL-8 were found in the medium micromolar range. No indication for increasing affinities for monomeric IL-8 with increasing oligosaccharide chain length was found. Instead, a periodic pattern was obtained for the dissociation constants of the GAG oligosaccharides with respect to chain length, referring to optimum and least optimum chain lengths for IL-8 binding. GAG disaccharides were identified to be the minimum length for chemokine binding. Conformational changes of the dimeric chemokine, determined using CD spectroscopy, were detected only for the IL-8/HS complexes and not for heparin, pointing to an HS-induced activation of the chemokine with respect to receptor binding. Thermal unfolding of IL-8 yielded a single transition at 56 degrees C which was completely prevented by the presence of undigested HS or heparin, indicating structural stabilization, thereby prolonging the biological effect of the chemokine.

The intrinsic tryptophan fluorescence signal of the full-length nuclear receptor hVDR was used to directly determine the dissociation constants, K d , of two ligands yielding K d = 32 nM for 1α,25(OH) 2 D 3 and K d = 322 nM for 25(OH)D 3 . Ligand binding was accompanied by a conformational change in the α-helical part of hVDR as revealed by CD spectroscopy. In addition, the presence of calcitriol was found to be a necessary prerequisite for the homodimerisation of hVDR which was monitored using fluorescence anisotropy. We conclude that the observed ligand-induced structural change of hVDR is conditional for dimerisation of the protein.

The secondary structure of the ligand response domain of the Haemophilus influenzae tyrosine repressor, TyrR(lrd), was investigated using CD spectroscopy which revealed 42.5% alpha-helix, 17.6% beta-sheet, and 39.9% loops. Quaternary structure analysis by fluorescence anisotropy showed that TyrR(lrd) is monomeric at a concentration of 100 nM to 2 microM but that the protein readily dimerizes in the presence of its natural ligand ATP. Equilibrium unfolding studies of TyrR(lrd) using guanidinium hydrochloride suggested a two-state model with no detectable stable intermediates. The unfolding transition monitored by CD spectroscopy was responsive to tyrosine and ATP resulting in a shift to higher denaturant concentrations in the presence of these ligands. Differential scanning calorimetry yielded melting temperatures, T(m), of 51.15 and 58.07 degrees C for the unliganded and for the ATP-liganded protein, respectively. ATP is thus proposed to be a major structural cofactor for the molecular architecture of TyrR(lrd).

The binding of aromatic amino acids to the ligand response domain of the tyrosine repressor (TyrR) protein (TyrRlrd) of Haemophilus influenzae was investigated using circular dichroism and fluorescence spectroscopy. The induced secondary structural changes were unique for each aromatic amino acid and were further influenced by the presence or absence of ATP. Tyrosine was found to have the highest affinity for TyrRlrd in the absence of ATP, whereas the affinity for ATP itself increased in the presence of tyrosine. Binding of tyrosine is therefore the conformational trigger for the activation of TyrR whereas ATP is regarded as a conformational co-activator.