Báo cáo khoa học: Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli potx

Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli Rositsa Jordanova 1 , Georgi Radoslavov 1 , Peter Fischer 2 , Eva Liebau 2 , Rolf D. Walter 2 , Ilia Bankov 1 and Raina Boteva 3 1 Institute of Experimental Pathology and Parasitology, Sofia, Bulgaria 2 Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany 3 National Center of Radiobiology and Radiation Protection, Sofia, Bulgaria Lipid-binding proteins (LBPs) regulate the physiological activity, metabolism and disposition of essential hydro- phobic compounds like fatty acids, phospholipids, eicosanoids and retinoids. Fatty acids and phospho- lipids are the major energy reserves and components of the cell membranes, whereas eicosanoids and retinoids are important signaling molecules involved in several cellular processes including gene transcription, cell growth and differentiation, tissue repair, inflamma- tion and immune responses. Conjugated with LBPs, Keywords NPA, Trp and IAEDANS fluorescence, FRET, immunohistology Correspondence R. Boteva, National Center of Radiobiology and Radiation Protection, Sofia 1756, Bulgaria Fax: +359 28621059 Tel: +359 28626036/210 E-mail: r.boteva@ncrrp.org (Received 27 July 2004, revised 17 September 2004, accepted 20 September 2004) doi:10.1111/j.1432-1033.2004.04398.x Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascari- dia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, a-helical protein and that ligand binding slightly increases the ordered sec- ondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromo- phores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emis- sion, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfo- nyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chem- ical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naph- thalene-1-sulfonic acid (IAEDANS) and N,N 0 -dimethyl-N-(iodoacetyl)-N 0 - (7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 sug- gest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids. Abbreviations DAUDA, 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid; FRET, fluorescence resonance energy transfer; IAEDANS, 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid; IANBD, N,N 0 -dimethyl-N-(iodoacetyl)-N 0 -(7-nitrobenz-2-oxa-1,3-diazol- 4-yl)ethylenediamine; LBP, lipid binding protein; NPA, nematode polyprotein allergens/antigen. 180 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS these compounds are solubilized, protected from chemical damage and delivered to the correct destina- tion [1–4]. LBPs from parasitic nematodes are of special inter- est because these organisms typically exhibit limited lipid metabolism and have to import complex lipids from the host [5]. Nematodes possess two classes of structurally novel types of helix-rich LBPs [6–8]. The first class consists of small 15 kDa fatty acid and reti- noid binding proteins, characterized by extremely non- polar binding sites. They are synthesized as large precursor polypeptides and subsequently cleaved into functional units. Based on this peculiarity and on their allergenicity, these LBPs are named nematode polyprotein allergens/antigens (NPAs) [6,9,10]. The second class of fatty acid and retinoid binding proteins (FAR proteins) are slightly larger, 20 kDa, with stron- ger affinity for retinol than for fatty acids. They nota- bly differ in their amino acid sequence from NPAs [11]. Ag-NPA-1 from the parasitic nematode Ascarida galli is a member of the NPAs family [12]. Its fatty acid and retinoid binding activities have been studied indirectly in displacement experiments with the fluores- cent substrate 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA), a dansyl- ated undecanoic acid. The fluorescence properties of DAUDA strongly depend on the polarity and accessi- bility of the protein binding sites and this has been widely used in studies on the binding properties of parasitic as well as of mammalian LBPs [9,12–16]. The changes in the dye emission upon binding characterize the binding sites of NPAs as highly nonpolar and com- pletely isolated from the solvent. Here we analyze the conformational and functional properties of Ag-NPA-1 as well as the tissue and cellular distribution of the protein. The binding affinities are characterized by changes in the fluorescence of the single Trp chromophore that is used as a marker of the protein conformation and of the thiol reactive probe 5-({[(2- iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (1,5-IAEDANS), covalently attached to Cys66. Results Conformational and oligomeric properties of Ag-NPA-1 After gel filtration, natural or recombinant Ag-NPA-1 was eluted in a single protein peak of  24 kDa sug- gesting a dimer formation. In several species, the units of the nematode polyproteins differ from each other in their amino acid sequences [7]. This, however, is probably not true for the group of nematodes to which A. galli belongs as suggested by the molecular homo- geneity of native Ag-NPA-1 proved by N-terminal sequencing followed by MS analysis (data not shown). Native gel electrophoresis in the presence and absence of palmitate, which is one of the preferred ligands of Ag-NPA-1 [12], showed that the binding did not cause any changes in the protein oligomeric state (data not shown). The pI of Ag-NPA-1 was determined by 2D gel electrophoresis and compared with the value calcu- lated from the protein amino acid sequence. A good correspondence of the experimentally determined pI of 6.1 and the theoretically deduced pI value of 6.22 was found. A theoretical prediction of the secondary structural organization of Ag-NPA-1 performed on the basis of the amino acid sequence [12] showed up to 80% a-helical content. The model of the backbone folding [17] suggests that the protein molecule is organized in four helices. The CD measurements in the far UV-region (190–260 nm) confirmed the theoretical prediction and showed that Ag-NPA-1 was a typical a-helical protein containing 66% a-helices and 12% b-turns (Fig. 1). Incubation of the protein with dif- ferent ligands such as palmitate, caprylic and arachi- donic acids and retinol in concentrations sufficient to saturate the protein binding sites, caused similar effects; slight enhancement of the helical content at the expense of random coil mainly. Helical content reached 78% in the presence of caprylic acid, indica- ting additional stabilization of the Ag-NPA-1 confor- mation upon ligand binding. Role of Cys residues for Ag-NPA-1 conformation and function According to the amino acid sequence, Ag-NPA-1 con- tains two Cys residues at positions 66 and 122 [12]. 200 225 250 -5000 0 5000 10000 λ nm [ θ ] R deg cm 2 dmol -1 Fig. 1. CD spectrum of Ag-NPA-1 in the far UV-range (190– 260 nm). R. Jordanova et al. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS 181 The theoretical modelling of the protein backbone folding predicts that these two residues are localized on two neighbouring helices and the separation between their C a atoms approaches 1 nm, a distance, suitable for a disulfide (S-S) bridge formation. The electrophoretic analysis of the native protein by gra- dient SDS/PAGE, performed in the presence and absence of the reducing agent 2-mercaptoethanol, showed no changes in the migration of the protein when free or conjugated with palmitate. This suggests that even if Cys66 and Cys122 formed a disulfide bridge, it is not important for the structural integrity and stability of the Ag-NPA-1 molecule. This was further tested by chemical modification of Ag-NPA-1 with two fluorescent iodacetamides, IAEDANS and IANBD, characterized by high specifi- city and reactivity to free sulfhydryl groups [18]. The covalent binding of the dyes to either native or recom- binant Ag-NPA-1 was confirmed by denaturation of the labeled proteins with 6 m guanidium chloride [19]. This procedure did not cause any release of the markers as both emission and absorbance bands specific to IAEDANS or IANBD could be registered. The quantity of the bound dye was determined spectrophoto- metrically and showed binding of one molecule of either IAEDANS or IANBD to a protein monomer. The labeled Cys residue was identified by MALDI- TOF analysis of the trypsin digested Ag-NPA-1 [20]. Cys66 and Cys122 were found in the peptide fragments of the nontreated, natural protein with theoretically calculated masses of 1033.5459 Da (AKESLIGGCR) and 1236.6292 Da (ELIKDYGPACK). However, in the mass spectrum of Ag-NPA-1 covalently labeled with IAEDANS or IANBD, the 1033.5459 Da frag- ment containing Cys66 could not be detected. This could be explained by the chemical modification of Cys66, leading to a change in the properties of the dye-carrying fragment which prevented its detection. Thus, Cys66 is the reactive and accessible to the bulky dye molecules residue. The fluorescence properties of IAEDANS are strongly dependent on the polarity of its environment. The emission maximum of the dye bound to Ag-NPA- 1 is at 460 nm, typical for a chromophore in a highly hydrophobic environment [19]. The exposure and accessibility of the dye attached to Cys66 were further characterized by acrylamide quenching. We calculated a Stern–Volmer constant (K Q ) of 6.7 m )1 which, when compared to the K Q of 14.8 m )1 for the free dye, indi- cated a partial accessibility of the marker to external solvent molecules. Binding of palmitate, identified as one of the preferred ligands [12], caused an additional 25–30 nm blue-shift of the emission maximum position of the dye accompanied by almost twofold emission intensity enhancement. These changes indicated significant conformational rear- rangements in Ag-NPA-1 molecules upon ligand bind- ing which strongly affected the surrounding of Cys66 and increased its hydrophobicity. The fluorescence changes allowed calculation of the apparent dissociation constant K d of the protein–palmitate complex. A value of 0.25 ± 0.10 lm, similar to that reported in [12] for the native protein was obtained. The similar affinities of the native and modified proteins suggest that Cys66 is not a part of the binding site, however, it is located close to the binding pocket as the ligand binding strongly influences the fluorescence properties of the dansyl chro- mophore, covalently attached to this Cys. Intrinsic fluorescence properties of Ag-NPA-1 The protein fluorescence emission spectrum obtained upon 275 nm excitation (where both Tyr and Trp chromophores absorb) shows a maximum at 318 nm (Fig. 2). Upon excitation at 300 nm, where only the Trp chromophore absorbs, the emission maximum was registered at 325 nm, a position, indicative of a highly hydrophobic environment of the single Trp residue. The contribution of Tyr fluorescence to the overall protein emission was calculated to amount to 45% and the Trp emission quantum yield 0.015. The low value of the quantum yield indicates a strong conformational quenching of the Trp chromophore. To further characterize the location and accessibility of Trp17, quenching experiments with acrylamide as 290 310 330 350 370 390 0 5 10 15 a b c λ nm fluorescence (a.u.) Fig. 2. Fluorescence emission spectra of Ag-NPA-1. Spectra ‘a’ and ‘b’ are obtained with 275 and 300 nm excitation, respectively. Spectrum ‘c’ represents the Tyr contribution and is calculated as a difference between spectra ‘a’ and ‘b’ after their normalization above 380 nm. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 R. Jordanova et al. 182 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS an external quencher were performed (Fig. 3). We calculated a low value of 1.3 m )1 for the K Q con- stant which indicated a poor accessibility of the sin- gle Trp residue to external solvent molecules by pointing out a position in the hydrophobic interior of the protein molecules. As the Trp absorption spectrum overlaps the Tyr emission, a radiation-less energy transfer from Tyr to Trp chromophores could take place. We studied this process and found a relatively high efficiency of  65% (Fig. 4) which suggests a high degree of Tyr to Trp dipole-dipole coupling. Binding activities of Ag-NPA-1 determined by changes in Trp fluorescence Binding of retinol, oleic and arachidonic acids caused a slight (£ 11%) increase of the emission of the single Trp residue of Ag-NPA-1. Saturation of the binding sites followed a hyperbolic trend (Fig. 5) and the Trp emission maximum remained at 325 nm. From the Trp fluorescence enhancement, we calculated values for K d of 0.30 ± 0.04 lm for retinol, 0.23 ± 0.10 lm for oleic and 0.15 ± 0.01 lm for arachidonic acid. These values were similar to those reported in [12] which were calculated from fluorescence displacement experi- ments with DAUDA where the fatty acids, retinoids and DAUDA competed for the single binding site of the Ag-NPA-1 monomers. FRET between Trp17 and DAUDA in Ag-NPA-1 The absorption spectrum of DAUDA (maximum at 335 nm) largely overlaps the protein Trp emission. Hence, a fluorescence radiation-less energy transfer (FRET) from the singlet excited state of Trp residues to the dansyl group of the bound fluorescent fatty acid could be envisaged. This process would result in quenching of protein Trp fluorescence and in an increase of the specific DAUDA emission after the noncovalent incorporation of the ligand. Binding of DAUDA to Ag-NPA-1 caused  20% quenching of the emission of Trp17 indicating FRET between the single Trp residue and the dansyl group. We calculated a value of 3.74 · 10 )15 cm 3 Æm )1 for the spectral integ- ral J AD and of 0.012 for the Trp emission quantum yield (Q Trp-A ) of the protein–DAUDA complexes. Hence a critical distance (R o ) of 1.05 nm and an aver- age intramolecular distance (r) of 1.41 nm, between Trp17 and the dansyl chromophore from the binding site were estimated. This supports previous observa- tions (A Timanova, EPP, Sofia, Bulgaria, unpublished data) and shows that like the Trp residue of the 0.00 0.05 0.10 0.15 1.0 1.1 1.2 1.3 [acrylamide] M F 0 /F Fig. 3. Quenching of Trp fluorescence of Ag-NPA-1 by acrylamide. A value of 1.3 M )1 was calculated for the K Q constant. 260 270 280 290 300 0.8 0.9 1.0 Φ trp /Φ 300 e=0.5 e=0.65 e=0.8 e=1 λ nm Fig. 4. Tyr to Trp energy transfer efficiency in Ag-NPA-1. The curves are theoretical and are obtained for different values of trans- fer efficiency e. The experimental data are represented by empty triangles (n). They follow best the theoretical curve with e ¼ 65%. 0.00 0.25 0.50 0.75 1.00 0.0 2.5 5.0 7.5 10.0 oleate [µM] ∆ F % Fig. 5. Binding of oleic acid, followed by the enhancement of the Trp fluorescence (DF). The curve fits best the experimental data, obtained with a K d constant of 0.23 ± 0.10 lM, calculated by a non- linear regression for a single binding site. R. Jordanova et al. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS 183 homologous ABA-1 [21], the single Trp of Ag-NPA-1 is not a part of the lipid binding pocket. Like DAUDA, the retinol absorption spectrum lar- gely overlaps Trp emission and FRET from the Trp residue, buried in the interior of the protein molecule, to the ionone ring of retinol would be expected. Inter- estingly, binding of retinol caused an increase of the emission of the single Trp residue, similar to that regis- tered after binding of oleic and arachidonic acids, indicating no dipole-dipole interactions between the two types of chromophores. FRET is exponentially dependent on the distance between the donor-acceptor pair [22]. Therefore, the process should be most effi- cient within the Fo ¨ rster’s radius of 1.5 nm which we calculated for the Trp-retinol couple in Ag-NPA-1. As no energy transfer could be detected after the forma- tion of the Ag-NPA-1–retinol complexes, either the distance between the Trp residue and retinol is signifi- cantly longer than 1.5 nm or there is an unfavourable mutual orientation of the chromophores for dipole- dipole interactions. Immunohistology and immunogold TEM Using the antiserum raised against native Ag-NPA-1, worms fixed either with ethanol or formalin were stained. In general, the labeling was more intense in eth- anol fixed specimens compared to the formalin fixed ones. The preimmune serum, used as a negative control, showed absence of unspecific reactions (Figs 6A and 7A). A significant staining of the fluid of the pseudocoe- lomatic cavity was observed (Fig. 6B). In A. galli the inner hypodermis (Fig. 6D), the lateral and the median chord were mainly stained. Furthermore, sperm that were attached to the uterus tissue and the oviduct, were intensively labeled in contrast to the ovary and the uterus which were not stained. No staining was also observed in the cuticle, the muscle syncytia or the intes- tine (Fig. 6B). Besides, the antibody, raised against Ag- NPA-1 gave cross-reactions with similar proteins from other ascaridis. When the localization of the protein in A. galli was compared to that in Ascaridia suum, a sim- ilar staining pattern was found (Fig. 6C,E,F). However, Fig. 6. Immunohistological localization of Ag-NPA-1 in adult A. galli and comparison with that in A. suum using a polyclonal anti- serum raised against native Ag-NPA-1. (A) Section of A. galli showing the ovary (ov) the uterus (ut) the intestine (i) and the parts of the pseudocoel (ps) stained with the pre- immune serum as control. (B) Consecutive section of A showing intense staining of the pseudocoel (ps) especially in the vicinity of the uterus (ut) with developing eggs. (C) Strong labeling of the oviduct (ovi) next to the unstained ovary (ov) in A. suum. (D) Section of the body wall of a female A. galli showing an intense labeling of the inner hypodermis (ihy, arrow). (E) Staining of the labyrinth of the lateral chord (lc, arrow) in A. suum. (F) Staining of the median chord (mc, arrow) in A. suum. Bar size is 50 lm (A–F). Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 R. Jordanova et al. 184 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS an equal intensity of staining was obtained in A. suum with a primary antiserum dilution of 1 : 1000 compared to 1 : 4000 in A. galli (Fig. 6). Ultrastructural localization of Ag-NPA-1 in A. galli by immunogold electron microscopy confirmed the results from the light microscopy. Sections through the contractile portion of the somatic musculature revealed that the interstitial space between the striate muscula- ture, which is filled with pseudocoelomatic fluid, was also strongly labeled by gold particles (Fig. 7B). These observations suggest that Ag-NPA-1 is localized mainly in cells of the inner hypodermis and the epithe- lium of the oviduct as well as extracellularly in the pseudocoelomic cavity of the worms. Discussion A bundle of four a-helices constitutes the secondary structure organization of Ag-NPA-1 and of other homologous NPAs as suggested by theoretical predic- tions of the protein backbone folding. These helices might shape the hydrophobic binding pocket which was shown to bind fatty acids, retinoids and arachi- donic acid with high affinity. CD analysis confirmed the predicted helical structure of Ag-NPA-1 and showed 66% a-helical content for both native and recombinant proteins. It increased 10–12% upon lig- and binding, suggesting additional conformational sta- bilization of the protein in the complexes. The single Trp and the two Cys residues are highly conserved in all amino acid sequences of NPAs from parasitic nematodes [12] suggesting important struc- tural or functional roles of these residues. According to secondary structure predictions, Trp17, Cys66 and Cys122 are part of three neighboring helices and the distance between the two Cys residues is suitable for S-S bridge formation. The chemical modification of Cys66 by two different, highly specific to free sulfhyd- ryl groups fluorescence dyes, IAEDANS and IANBD, shows that this Cys residue is not involved in disulfide bonding. According to the emission characteristics of the covalently attached dyes, Cys66 is located in a hydrophobic environment and is partially accessible to external solvent molecules. In spite of its high reactiv- ity Cys66 is not a part of the protein binding pocket as K d similar for the palmitate binding by the native and by the chemically modified Ag-NPA-1 were found [12]. The ligand binding significantly increased the IAEDANS emission and caused 20 nm blue-shift of the emission maximum, changes indicating significant rear- rangements in the protein molecule which increased the hydrophobicity of the Cys66 environment. In contrast to the homologous ABA-1 protein whose emission spectrum was strongly dominated by Tyr fluor- escence and the Trp contribution was registered as a shoulder [21], only the Trp fluorescence of Ag-NPA-1 showed a peak with a maximum at 325 nm, typical for a chromophore in a highly nonpolar environment. These differences in the emission properties of the proteins probably reflect local conformational differences between the two homologous NPAs. Trp17 is deeply buried in the hydrophobic interior of the Ag-NPA-1 molecule and poorly accessible to the solvent as sugges- ted by the very low value of the quenching constant (K Q 1.3 m )1 ) obtained with acrylamide as external quencher. The contribution of Tyr chromophores to the overall protein fluorescence was  45% and the efficiency of Tyr to Trp energy transfer 65%, suggesting a high degree of Tyr-Trp dipole-dipole coupling. The Trp fluorescence increased up to 11% upon binding of fatty acids, retinol and DAUDA that allowed studies on the protein binding affinities. The values of the dissociation constants calculated by chan- ges in Trp emission were close to those determined in displacement experiments with the fluorescent fatty acid analogue DAUDA [12]. Thus, in contrast to the homologous Trp chromophore of ABA-1, the single Trp of Ag-NPA-1 is a sensitive marker of the protein conformation and its emission reflects conformational changes after ligand binding. Analysis of FRET from the singlet excited state of Trp17 to DAUDA, noncova- lently bound to Ag-NPA-1, allowed calculation of the Fig. 7. Immunogold electron microscopic localization of Ag-NPA-1 in a male A. galli worm. (A) Section of the striate musculature (mu) and the interstitial space (is) stained with the preimmune serum as primary antibody. (B) Consecutive section to A using the antiserum raised against native Ag-NPA-1 showing strong accumulation of gold particles in the pseudocoelomatic fluid of the interstitial space (arrows). Bar size is 0.5 lm (A–B). R. Jordanova et al. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS 185 average distance between these chromophores. The dis- tance approaches 1.41 nm which suggests that Trp17 of Ag-NPA-1, like the single Trp residue of ABA-1 [21], is not involved directly in the protein binding pocket. The first described NPA was ABA-1 from A. suum. It is an allergen from the excretory-secretory (ES) products of the nematode and has a high affinity for fatty acids and retinoids [8]. This finding applies to all the representatives of the family and suggests an important role of these proteins in importing essential lipids from the host. Furthermore, worms could use this mechanism to export hydrophobic signalling mole- cules, including retinoids, in order to modulate the host response. By reporter-gene assays in Caenorhabdi- tis elegans [7] and by Northern blotting in Ascaris [23], the cells of the gut were identified as a place of the NPA synthesis. The immunohistological analysis in this study suggests that Ag-NPA-1 is distributed mainly in the pseudocoelom of A. galli. This indicates an additional function of the protein as an internal transporter of lipid metabolites in the parasitic tissues. Cross reactions of the Ab used for the comparative localization in A. suum and Anisakis larvae confirmed this distribution. Besides, in Anisakis a secretory cell was specifically labeled indicating a possible mechan- ism of protein excretion in the host tissues (data not shown). However, as no similar structure could be identified in A. galli and A. suum, no comparison was possible. Interestingly, the localization of Ag-NPA-1 to the hypodermis is similar to that of the lipid binding proteins in the filarial parasites of humans [24,25] which lends them to in situ iodination in the whole living worms. EM experiments localized the protein mainly extracellularly, in the interstitial space, but also in some of the muscle cells. Although a specific cell surface receptor could exist it is possible that this small protein interacts directly with the cell membranes, as reported for ABA-1 [26], and acts as a shuttle for delivering lipids to the place of their metabolic trans- formation. In summary, the general distribution of Ag- NPA-1, which is one of the main proteins in A. galli cytosol, suggests important functions of the protein in the internal lipid transport, which might be essential for the parasite survival as these organisms exhibit lim- ited ability to synthesize long chain fatty acids de novo. Experimental procedures Protein purification Natural and recombinant Ag-NPA-1 from A. galli used in the experiments were purified as described previously [12,16]. The protein concentration was determined spectro- photometrically using a molar extinction coefficient of 9.5 · 10 3 m )1 Æcm )1 at 280 nm as calculated on the basis of the aromatic amino acid content of one Trp and three Tyr residues per protein monomer [27]. Fluorescence measurements and reagents The fluorescent fatty acid analogue 11-[(5-dimethylamino- naphthalene-1-sulfonyl) amino] undecanoic acid (DAUDA) and the thiol reactive probes 5-({[(2-iodoacetyl) amino]}eth- ylamino) naphthalene-1-sulfonic acid (1,5-IAEDANS) and N,N 0 -dimethyl-N-(iodoacetyl)-N 0 -(7-nitrobenz-2-oxa-1,3-dia- zol-4-yl)ethylenediamine (IANBD) were obtained from Molecular Probes (Eugene, OR, USA). Retinol and fatty acids were obtained from Sigma (St Louis, MO, USA). IAEDANS and IANBD were dissolved in N,N-dimethyl formamide, the fatty acids and retinol in ethanol at concen- trations of 1 or 0.1 mm. The concentrations of DAUDA, IAEDANS, IANBD and retinol were calculated from their absorption spectra using the corresponding molar extinc- tion coefficients. The concentration of the organic solvents in the final reaction did not exceed 2%. Steady-state fluorescence was measured with a Shimadzu model RF5000 spectrofluorometer and a Kontron SMF 25, both equipped with thermostatically controlled cell holders. The relative Trp emission quantum yield (Q Trp ) was deter- mined by comparing the integrated fluorescence spectrum of the protein excited at 300 nm with that of the standard N-Ac-Trp-NH 2 normalized to the same absorbance at 300 nm. A value of 0.13 was used for the quantum yield of the standard [28]. In order to minimize inner filter and self- absorption effects, the sample absorbance at the excitation wavelength (k exc ) was always lower than 0.05. The effi- ciency of Tyr to Trp energy transfer was calculated by a procedure described in [29]. The Tyr contribution to the total protein fluorescence was estimated by subtraction of the Trp emission spectrum (k exc 300 nm) from that obtained at k exc 275 nm, after normalizing the two spectra above 380 nm, where the Tyr emission is neg- ligible. Quenching of Trp fluorescence and of the emission of the bound IAEDANS was performed with acrylamide as external quencher. The data were analyzed according to the Stern–Volmer equation [28]: F o /F ¼ 1+K Q [X], where, F o and F are the fluorescence emission intensities in the absence and presence of acrylamide, [X] is the molar concentration of acrylamide and K Q the overall quenching constant. Circular dichroism measurements CD spectra were recorded in 10 mm Tris, pH 7.5, 20 °C, using a Jasco Model 715 automatic recording circular dichroism spectrophotometer with a thermostatically con- trolled cell holder. A fused quartz cell with a path-length of 0.1 cm was used. The protein concentration was 0.82 mm. The spectra measured in the far UV-region 190–260 nm Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 R. Jordanova et al. 186 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS were averages of four scans and were corrected by subtract- ing the baseline of the buffer. They are reported as mean residue molar ellipticity ([h] R ) in degrees cm 2 Ædmol )1 . Spec- tra subtraction, normalization and smoothing were per- formed by using jasco cd j-715 data manipulation software and the analysis of the data was carried out with the programs contin and selcon. Protein labeling Ag-NPA-1 was covalently labeled with the thiol reactive probes 1,5-IAEDANS and IANBD. Labeling was per- formed with the native and the recombinant proteins in 10 mm Tris, pH 7.4 after incubation for 4 h at 4 °C with a 20-fold molar excess of the dyes. The unbound dye was removed by gel filtration with Bio-Spin30 Tris columns (Bio-Rad). The extent of labeling and the protein to dye ratio were determined spectrophotometrically, from the protein absorbance at 280 nm (e M,280 9.5 · 10 3 M )1 Æcm )1 for Ag-NPA-1) and the dye absorbances at 337 nm for IAEDANS (e M,337 6 · 10 3 M )1 Æcm )1 ) and at 472 nm for IANBD (e M,472 23 · 10 3 M )1 Æcm )1 ). The covalent attach- ment of the dyes was confirmed after denaturation of the labeled protein with 6 m guanidine hydrochloride followed by dialysis against 3 m guanidine hydrochloride in 10 mm Tris buffer, pH 7.5. Then, both the absorption and emis- sion spectra were recorded. In addition to the protein peaks, they also contained IAEDANS or IANBD bands, indicative of covalent binding of the dyes [19]. Binding activities of Ag-NPA-1 Binding of fatty acids, retinol and DAUDA were studied by changes in the intrinsic Trp fluorescence of Ag-NPA-1 after excitation at 300 nm (k exc 300 nm). Ag-NPA-1 (0.2 lm) was incubated with increasing ligand concentra- tions overnight, 4 °C, in the dark, in order to prevent chemical changes of the light-sensitive ligands. As DAUDA and retinol absorb at the excitation (k exc 300 nm) and emis- sion (k em 325 nm) wavelengths, the emission spectra were corrected for inner filter effects and background fluores- cence [19]. A least-square analysis of the transformed data was carried out by Graphpad prism computer program. This allowed calculation of the apparent dissociation con- stants (K d ) and the maximal fluorescence change (DF max ) after a full saturation of the protein binding sites. Changes in the specific emission of the fluorescent probe IAEDANS (k exc 360 nm), covalently attached to Cys66 of Ag-NPA-1 upon binding of palmitate, were also examined. Fluorescence resonance energy transfer Intramolecular fluorescence resonance energy transfer (FRET) from the single Trp residue of Ag-NPA-1 (donor) to the dansyl group of DAUDA (acceptor) was studied by the decrease in the Trp fluorescence after saturation of the pro- tein binding sites with the fluorescent probe. This allowed calculation of the average distance r between the energy donor-acceptor pair: r ¼ R o [(1 ) E)/E] 1/6 , where, E is the efficiency of the energy transfer process, calculated from the decrease of the donor quantum yield (Q Trp ) in the presence of the acceptor (Q Trp-A ): E ¼ 1–Q Trp-A /Q Trp , where, R o is the Fo ¨ rster radius or the critical distance for a 50% probabil- ity of the energy transfer process: R o ¼ (9.79 · 10 3 ) · (J AD n )4 K 2 Q Trp ) 1/6 A ˚ , where J AD is the overlap integral between the decadic molar absorbance of the acceptor and the correc- ted emission spectrum of the donor on a wavenumber scale normalized to unity [30], n the refractive index of the medium and K 2 the orientation factor, determined by the mutual spa- tial orientation of the transition dipole moments of the donor and acceptor. As no data on the spatial orientation of the transition dipole moments of the chromophores are avail- able, a random orientation of the donor–acceptor pair was assumed (K 2 0.667 [30]). A value of 1.36 was taken for the refractive index n [31]. MALDI-TOF analysis MALDI-TOF analysis of the peptides obtained after tryptic digestion of the labeled and nonlabeled Ag-NPA-1 was per- formed as described in [20,32]. The data were analyzed with peptide mass software (us.expasy.org/tools). The chemically modified Cys residue was identified indirectly, upon com- parative analysis of the peptide patterns obtained after proteolytic cleveage of the Ag-NPA-1 with and without treatment by the sulfhydryl reagents. Data analysis and structure predictions Sequence analysis and secondary structure predictions were performed with programs available on the ExPaSy mole- cular biology server (us.expasy.org/tools/); the molecular mass and isoelectric point (pI) of the protein were estimated with the protparam program; goriv and jpred programs were used for secondary structure predictions and 3d-pssm software for backbone fold recognition [17]. Immunohistology and immunogold TEM An antiserum against Ag-NPA-1 was raised in a rabbit using a standard immunization protocol (Eurogentec, Sera- ing, Belgium). The preimmune serum was used as a con- trol. Immunolocalization on the light and on the electron microscopical level was performed as described previously [33]. For light microscopic immunohistology, adult A. galli worms were either fixed in 4% (v/v) buffered formaldehyde or in 80% (v/v) ethanol and embedded in paraffin. For R. Jordanova et al. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS 187 comparison with other ascaridis sections of female A. suum were also used. The alkaline phosphatase-antialkaline phos- phatase (APAAP) technique was used for immunostaining according to the manufacturer’s recommendations (Dako Diagnostika, Hamburg, Germany). As primary antibodies, the rabbit antitserum against Ag-NPA-1 or the preimmune serum were used at dilutions of 1 : 500 to 1 : 4000. As sec- ondary antibodies, anti-rabbit, mouse immunoglobulins (Dako) were applied and Fast Red TR salt (Sigma, Dei- senhofen, Germany) was used as chromogen. Hematoxylin functioned as the counterstain. For immunogold TEM, adult A. galli worms were fixed for 4 h in a solution of 1% paraformaldehyde and 0.025% glutardialdehyde. Then samples were preserved in 0.2 m sodium cacodylate buffer (pH 7.2) and stored at 4 °C. Dehydrated specimens were embedded in medium-hardness LR white acrylic resin (Polysciences, Warrington, USA). Ultrathin sections were collected on filmed hexagonal nickel grids (200 mesh). Following incubation in NaCl/P i , samples were blocked in 10% BSA and incubated with the primary antiserum at a dilution 1: 10000. After washing, the sec- tions were treated with Protein A Gold 10 nm (University of Utrecht, School of Medicine, Department Cell Biology, NL) at a dilution of 1 : 70. Later, the sections were fixed in 2% glutardialdehyde and counterstained as described above. For negative controls, the primary antibody was replaced by the corresponding preimmune sera. Acknowledgements R.J. was supported by Deutscher Academischer Austauschdienst (DAAD) and Deutsche Forschungs- gemeinschaft (DFG). 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Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli Rositsa Jordanova 1 ,. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS 181 The theoretical modelling of the protein