A modification of neurotoxin RTX-III from Radianthus macrodactylus actinin with acetic anhydride

Upon modification of neurotoxin RTX-III at amino groups with [3H]acetic anhydride, four monoacetyl and four diacetyl derivatives have been obtained. Acetylation of the N-terminal amino group led to 12-fold decrease of toxicity in mice. Monoderivatives of the toxin with either Lys or two out of three C-terminal Lys residues modified showed a 2-fold drop in toxicity as compared with the native RTX-III. Diacetylation caused a 30 to 35-fold decrease in toxicity, the N-terminal amino group being modified in all the derivatives. As assessed by circular dichroism method, the modification of amino groups, except for N-terminal one, affected secondary structure of the toxin. The data suggest the N-terminal amino group to be essential for toxicity of RTX-III.     

Actions between neonicotinoids and key residues of insect nAChR based on an ab initio quantum chemistry study: hydrogen bonding and cooperative pi-pi interaction

Neonicotinoid insecticides show selective actions on insect nicotinic acetylcholine receptor (nAChR). Two key residues (Trp and Arg/Lys) have been identified as contributing to the neonicotinois binding. To investigate the selective mechanism, a computational model was set up to simulate the interaction between residues (Trp and Arg) of insect nAChR and neonicotinoids by quantum chemistry method. Three analogues of neonicotinoid derivatives without the chloropyridinyl moiety and 3-methyl-indole (3MI), guanidinium (Gua) were used to mimic the neonicotinoids and the side chain of key residues Trp and Arg accordingly. Interaction features of 3MI-analogues, analogues-Gua and 3MI-analogues -Gua complexes were analyzed comparatively. Hydrogen bonding between the nitro group of analogues and Gua was found to be the most important for binding. Moreover, the cooperative pi-pi interaction between analogues and the indole ring, which is strengthened by the existence of Gua, also contributes to the binding. The alternative binding model of neonicotinoids proposed here, although slightly different from others, might be close to the actual.     

Deciphering the structural elements of hirudin C-terminal peptide that bind to the fibrinogen recognition site of alpha-thrombin

The C-terminal peptide of a hirudin acts as an anticoagulant by binding specifically to a noncatalytic (fibrinogen recognition) site of thrombin. This binding has been shown to shield five spatially distant lysines of the thrombin B-chain (Lys21, Lys65, Lys77, Lys106, and Lys107). It was also demonstrated that modification of the sequence of the hirudin C-terminal peptide invariably diminished its anticoagulant activity. The major object of this study is to investigate how the decreased activity of the modified hirudin C-terminal peptide is reflected by the change of its binding properties to these five lysines of thrombin. A synthetic peptide representing the last 12 C-terminal amino acids of hirudin (Hir54-65) was (1) truncated from both its N-terminal and its C-terminal ends, or (2) substituted with Gly along residues 57-62, or (3) chemically modified to add (sulfation at Tyr63) or abolish (Asp and Glu modification with carbodiimide/glycinamide) its negatively charged side chains. The binding characteristics of these peptides to thrombin were investigated by chemical methods, and their corresponding anticoagulant activities were studied. Our results demonstrated the following: (1) the anticoagulant activities of hirudin C-terminal peptides were quantitatively related to their abilities to shield the five identified lysines of thrombin. The most potent peptide was sulfated Hir54-65 (S-Hir54-65) with an average binding affinity to the five lysines of 120 nM. A heptapeptide (Hir54-60) also displayed anticoagulant activity and thrombin binding ability at micromolar concentrations. (2) All active hirudin C-terminal peptides regardless of their sizes and potencies were shown to be capable of shielding the five lysines of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

E. coli RNase HI and the phosphonate-DNA/RNA hybrid: molecular dynamics simulations

A model for the complex between E. coli RNase HI and the DNA/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3-O-CH2-P-O-5' or 3-O-P-CH2-O-5) on the ability of the modified-DNA/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to the 3-end of the DNA strand to interfere with the DNA binding site of RNase H. Altogether, six trajectories were produced (length 1.5ns). Mutual hydrogen bonds connecting both strands of the nucleic acids hybrid, DNA with RNase H, RNA with RNase H, and the scissile bond with the Mg++. 4H2O chelate complex (bound in the active site) were analyzed in detaiL Many residues were involved in binding of the DNA (Arg88, Asn84, Trp85, Trp104, Tyr73, Lys99, Asn100, Thr43, and Asn 16) and RNA (Gln76, Gln72, Tyr73, Lys122, Glu48, Asn44, and Cys13) strand to the substrate-binding site of the RNase H enzyme. The most remarkable disturbance of the hydrogen bonding net was observed for structures with modified internucleotide linkages positioned in a way to interact with the Trp104, Tyr73, Lys99, and Asn100 residues (situated in the middle of the DNA binding site, where a cluster of Trp residues forms a rigid core of the protein structure).     

Structure-activity relationships of endothelin: importance of the C-terminal moiety

The vasoconstrictor activities of various forms of derivatives of endothelin (ET) were characterized in vitro by measuring the contraction of porcine coronary artery strips. The removal of the C-terminal Trp21 reduced the molar potency of the peptide by nearly 3 orders of magnitude. The removal of amino acid residues from the C-terminus of ET(1-20) further attenuated the activity. Replacement of Trp21 with D-Trp, reduction and carboxamidomethylation of the four Cys residues, or cleavage at Lys9 by lysyl endopeptidase all lowered the potency approximately 200 fold. While both native ET and [D-Trp21]ET induced a very slow and sustained vasoconstriction, the other derivatives of ET listed above showed a much more rapid kinetics of vasoconstriction. These results indicate that the C-terminal Trp of ET is especially important for the potent and extremely long-lasting vasoconstrictor activity characteristic to ET.     

2′-N-Acetylation of Butirosin A by Mutants of Streptomyces fradiae

To reveal the mechanism of reducing sugar-induced polymerization of proteins, monomeric lysozymes were isolated at various stages of storage from whole lysozyme (WL) being kept with glucose at 75% relative humidity and 50°C for up to 30 days, and their chemical properties were investigated and compared with the corresponding WL. The impairment of Lys, Arg, and Trp residues was observed in all the isolated monomeric lysozymes (IM) as well as in the WL.

When the IM were stored for another 10 days without glucose, they polymerized and had an additional impairment of Arg and Lys but not Trp residues. All IM exhibited almost the same polymerization rate, but the sum of additional losses of Lys and Arg residues varied. The IM was also found to cross-link untreated lysozymes even in the absence of glucose.

On basis of the results obtained hitherto, it is suggested that the glucose-induced polymerization of lysozymes proceeds through the following paths. At the first step, some bifunctional agents (BF), probably α-dicarbonyl compounds, generated from the reaction between ?-amino groups of lysine residues and glucose, attach to Arg, Lys, and Trp residues through one of their two functional sites. At the second step, some of those proteins with BF attached polymerize by binding of the other unoccupied functional site with the remaining Lys and Arg (not Trp) residues of the other protein molecules. The other of the proteins with BF attached polymerize through the combination between the other unoccupied functional sites themselves with no loss of amino acid residues.     

Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites

Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.     

Cross Talk between KGF and KITLG Proteins Implicated with Ovarian Folliculogenesis in Buffalo Bubalus bubalis

Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.     

Inhibition of the binding and activation of the first component of human complement. The effect of synthetic peptides, immunoglobulin fragments and various proteins

A study has been carried out on the inhibition of the subcomponent Clq binding to sensitized sheep erythrocytes (EA) by the following synthetic peptides mimicking the structure of a putative complement binding site of immunoglobulin G: Boc-Trp-Tyr, Boc-Tyr-Trp, Trp-Tyr, Boc-Trp-Phe, Boc-D-Trp-D-Tyr, Boc-D-Tyr-D-Trp, Boc-Leu-Leu, Ac-Phe-Tyr, and commercial Thr-Lys-Pro-Arg (tuftsin). Boc-Trp-Tyr was found to be the most potent inhibitor of Clq binding to EA (Ki 2.86 X 10(-4) M), tuftsin ranking second with Ki 6 X 10(-4) M. The D,D-dipeptides failed to inhibit the Clq binding at the investigated concentrations. Insoluble Z-Trp-Tyr-OMe activated a classical pathway of complement system, as monitored by consumption of C4, C2 and C3 components. Synthetic octapeptide Boc-Glu-Val-Asp-Leu-Leu-Lys-Asp-Glu-OMe (corresponding to the sequence 36-43 of beta 2-microglobulin) inhibited the Clq binding with Ki 4.7 X 10(-4) M, which gave grounds for localizing the complement binding site in beta 2-microglobulin. The finding in the Clq structure of the peptide sequence homologous to than of the pepsin active site, as well as the close similarity in the specificity of these proteins towards hydrophobic amino acid residues justified the assumption on the same structural bases of their specificity. The results of the present study, along with the literature data, underlie the hypothesis on the involvement in the complement binding of the following IgG residues: Trp277, Tyr278, Lys320, Lys322, Glu318 and Lys290. The enlisted residues are closely located in the three-dimensional structure of the CH2 domain of IgG. Lysozyme and lactalbumin having the sequences homologous to Trp277-Tyr278 of IgG inhibited Clq binding to EA with Ki 3 and 1.5 microM respectively.     

Investigations on C-H...pi interactions in RNA binding proteins

We have investigated the roles played by C-Hcdots, three dots, centeredpi interactions in RNA binding proteins. There was an average of 55 C-Hcdots, three dots, centeredpi interactions per protein and also there was an average of one significant C-Hcdots, three dots, centeredpi interaction for every nine residues in the 59 RNA binding proteins studied. Main-chain to side-chain C-Hcdots, three dots, centeredpi interactions is the predominant type of interactions in RNA binding proteins. The donor atom contribution to C-Hcdots, three dots, centeredpi interactions was mainly from Phe, Tyr, Trp, Pro, Gly, Lys, His and Ala residues. The acceptor atom contribution to main-chain to side-chain C-Hcdots, three dots, centeredpi and side-chain to side-chain C-Hcdots, three dots, centeredpi interactions was mainly from Phe and Tyr residues. On the contrary, the acceptor atoms of Trp residues contributed to all the four types of C-Hcdots, three dots, centeredpi interactions. Also, Trp contributed both donor and acceptor atoms in main-chain to side-chain, main-chain to side-chain five-member aromatic ring and side-chain to side-chain C-Hcdots, three dots, centeredpi interactions. The secondary structure preference analysis of C-Hcdots, three dots, centeredpi interacting residues showed that, Arg, Gln, Glu, His, Ile, Leu, Lys, Met, Phe and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Trp and Val preferred to be in strand conformation. Long-range C-Hcdots, three dots, centeredpi interactions are the predominant type of interactions in RNA binding proteins. More than 50% of C-Hcdots, three dots, centeredpi interacting residues had a higher conservation score. Significant percentage of C-Hcdots, three dots, centeredpi interacting residues had one or more stabilization centers. Seven percent of the theoretically predicted stabilizing residues were also involved in C-Hcdots, three dots, centeredpi interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

pH-induced structural change of a multi-tryptophan protein MPT63 with immunoglobulin-like fold: identification of perturbed tryptophan residue/residues

The structural change of M. tuberculosis MPT63, which is predominantly a β-sheet protein having an immunoglobulin like fold, has been investigated in the pH range 7.5–1.5 using various biophysical techniques along with low-temperature phosphorescence (LTP) spectroscopy. MPT63 contains four Tryptophan (Trp) residues at 26, 48, 82, and 129. Although circular dichroism, steady-state and time-resolved fluorescence, time-resolved anisotropy, 1-aniline-8-naphthalene sulfonic (ANS) acid binding, and analytical ultracentrifuge depict more open largely unfolded structure of MPT63 at pH 1.5 and also more accessible nature of Trp residues to neutral quencher at pH 1.5, it is, however, not possible to assign the specific Trp residue/residues being perturbed. This problem has been resolved using LTP of MPT63, which shows optically resolved four distinct (0, 0) bands corresponding to four Trp residues in the pH range 7.5–3.0. LTP at pH 1.5 clearly reveals that the solvent-exposed Trp 82 and the almost buried Trp 129 are specifically affected compared with Trp 48 and Trp 26. Lys 8 and Lys 27 are predicted to affect Trp 129. Tyrosine residues are found to be silent even at pH 1.5. This type of specific perturbation in a multi-Trp protein has not been addressed before. LTP further indicates that partially exposed Trp 48 is preferentially quenched by acrylamide compared with other Trp residues at both pH 7.5 and 1.5. The solvent-exposed Trp 82 is surprisingly found to be not quenched by acrylamide at pH 7.5. All the results are obtained using micromolar concentration of protein and without using any Trp-substituted mutant.     

Structural/functional characterization of the alpha 2-plasmin inhibitor C-terminal peptide

The alpha(2)-plasmin inhibitor (A2PI) is a main physiological regulator of the trypsin-like serine proteinase plasmin. It is composed of an N-terminal 15 amino acid fibrin cross-linking polypeptide, a 382-residue serpin domain, and a flexible C-terminal segment. The latter, peptide Asn(398)-Lys(452), and its Lys452Ala mutant were expressed as recombinant proteins in Escherichia coli (r-A2PIC and r-A2PICmut, respectively). CD and NMR analyses indicate that r-A2PIC is flexible, loosely folded, and with low content of regular secondary structure. Functional characterization via intrinsic fluorescence ligand titrations shows that r-A2PIC interacts with the isolated plasminogen kringle 1 (r-K1) (K(a) approximately 69.9 mM(-)(1)), K4 (K(a) approximately 45.7 mM(-)(1)), K5 (K(a) approximately 4.3 mM(-)(1)), and r-K2 (K(a) approximately 3.2 mM(-)(1)), all of which are known to exhibit lysine-binding capability. The affinities of these kringles for r-A2PIC are consistently larger than those reported for the ligand N(alpha)-acetyllysine, a mimic of a C-terminal Lys residue. The r-A2PICmut, with a C-terminal Ala residue, also interacts with r-K1 and K4, although with approximately 5-fold lesser affinities relative to r-A2PIC, demonstrating that while Lys(452) plays a major role in the binding, internal residues in r-A2PIC tether the kringles. (1)H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp(25), Trp(62), Phe(64), Trp(72), and Tyr(74), selectively respond to the addition of r-A2PIC and r-A2PICmut, indicating that these interactions proceed via the kringles' canonical LBS. We conclude that r-A2PIC docks to kringles primarily through lysine side chains and that Lys(452) most definitely enhances the binding. This suggests that multiple Lys residues within A2PI could contribute, perhaps in a zipper-like fashion, to its binding to the in-tandem, multikringle array that configures the plasmin heavy chain.     

Anchoring mechanisms of membrane-associated M13 major coat protein

Bacteriophage M13 major coat protein is extensively used as a biophysical, biochemical, and molecular biology reference system for studying membrane proteins. The protein has several elements that control its position and orientation in a lipid bilayer. The N-terminus is dominated by the presence of negatively charged amino acid residues (Glu2, Asp4, and Asp5), which will always try to extend into the aqueous phase and therefore act as a hydrophilic anchor. The amphipathic and the hydrophobic transmembrane part contain the most important hydrophobic anchoring elements. In addition there are specific aromatic and charged amino acid residues in these domains (Phe 11, Tyr21, Tyr24, Trp26, Phe42, Phe45, Lys40, Lys43, and Lys44) that fine-tune the association of the protein to the lipid bilayer. The interfacial Tyr residues are important recognition elements for precise protein positioning, a function that cannot be performed optimally by residues with an aliphatic character. The Trp26 anchor is not very strong: depending on the context, the tryptophan residue may move in or out of the membrane. On the other hand, Lys residues and Phe residues at the C-terminus of the protein act in a unique concerted action to strongly anchor the protein in the lipid bilayer.     

E. COLI RNASE HI AND THE PHOSPHONATE-DNA/RNA HYBRID: MOLECULAR DYNAMICS SIMULATIONS

A model for the complex between E. coli RNase HI and the DNA/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3′–O–CH₂–P–O–5′ or 3′–O–P–CH₂–O–5′) on the ability of the modified-DNA/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to the 3′-end of the DNA strand to interfere with the DNA binding site of RNase H. Altogether, six trajectories were produced (length 1.5). Mutual hydrogen bonds connecting both strands of the nucleic acids hybrid, DNA with RNase H, RNA with RNase H, and the scissile bond with the Mg⁺⁺ · 4H₂O chelate complex (bound in the active site) were analyzed in detail. Many residues were involved in binding of the DNA (Arg88, Asn84, Trp85, Trp104, Tyr73, Lys99, Asn100, Thr43, and Asn16) and RNA (Gln76, Gln72, Tyr73, Lys122, Glu48, Asn44, and Cys13) strand to the substrate-binding site of the RNase H enzyme. The most remarkable disturbance of the hydrogen bonding net was observed for structures with modified internucleotide linkages positioned in a way to interact with the Trp104, Tyr73, Lys99, and Asn100 residues (situated in the middle of the DNA binding site, where a cluster of Trp residues forms a rigid core of the protein structure).     

Structural basis for the biological activity of dendrotoxin-I, a potent potassium channel blocker

A topochemical model to explain the biological activity of dendrotoxin-I (DTX-I), a potent blocker for potassium channels, was developed by searching common spatial arrangements of functionally important residues between DTX-I, alpha-dendrotoxin, dendrotoxin-K, BgK, ShK, and charybdotoxin. The first three are structurally and functionally related to one another, and specifically target to Kv1 type potassium channels. The last three are structurally unrelated to the first three but have the ability to displace (125)I-labeled dendrotoxins on the same types of potassium channels. In order to obtain the correct electronic surface potential, thought to be crucial for the DTX-I function, we determined the three-dimensional solution structure of DTX-I by nmr spectroscopy using its correct amino acid sequence recently determined by our group. The most interesting characteristic of our model is that DTX-I has two binding sites to potassium channels: one is the cationic domain made up of Lys residues at positions 5 in the 3(10)-helix, 28 and 29 in the beta-turn, and the other is the Lys19/Tyr17/Trp37 triad located in the antiprotease domain. The cationic domain and the triad are located at the opposite sides of the molecular structure and are separated by about 25 A between Lys29 Calpha and Tyr17 Calpha. The functional triad is characterized by three distances, d(1) approximately 7.5 A (Lys19 Calpha-the center of the Tyr17 aromatic ring), d(2) approximately 8.1 A (Lys19 Calpha-the center of the 6-membered ring of the Trp37 indole group), and d(3) approximately 7. 3 A (the center of the Tyr17 aromatic ring-the center of the 6-membered ring of the Trp37 indole group). This model should aid in the pharmaceutical design of peptide and nonpeptide drugs with potassium channel blocking potencies, as well as in understanding of the physiology, pharmacology, biochemistry, and structure-function analysis of potassium channels.     

An internal histidine residue from the bacterial surface protein, PAM, mediates its binding to the kringle-2 domain of human plasminogen.

The determinants of binding of a peptide lacking C-termini-exposed lysine residues to a kringle domain were investigated using an up-regulated lysine binding kringle (K2Pg[C4G/E56D/K72Y]) of plasminogen and a peptide (a1-PAM) with a sequence derived from a surface-exposed M-like streptococcal protein. Significant kringle-induced chemical shifts in a His side-chain of a1-PAM were revealed by two-dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His12 in the peptide/ protein complex. In an effort to screen a1-PAM-derived truncation peptides, a combinatorial mixture, a1deltaa2-PAM[H12X] (where X=Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2Pg[C4G/ E56D/K72Y]-containing chip was that containing His12, corresponding to the wild-type sequence. Minor peaks, representing binding, were obtained for Lys12-, Arg12- and Trp12-containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI-based screen.     

The role of tryptophan residues in the 5-Hydroxytryptamine(3) receptor ligand binding domain

Aromatic amino acids are important components of the ligand binding site in the Cys loop family of ligand-gated ion channels. To examine the role of tryptophan residues in the ligand binding domain of the 5-hydroxytryptamine(3) (5-HT(3)) receptor, we used site-directed mutagenesis to change each of the eight N-terminal tryptophan residues in the 5-HT(3A) receptor subunit to tyrosine or serine. The mutants were expressed as homomeric 5-HT(3A) receptors in HEK293 cells and analyzed with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Trp(90), Trp(183), and Trp(195) to tyrosine resulted in functional receptors, although with increased EC(50) values (2-92-fold) to 5-HT(3) receptor agonists. Changing these residues to serine either ablated function (Trp(90) and Trp(183)) or resulted in a further increase in EC(50) (Trp(195)). Mutation of residue Trp(60) had no effect on ligand binding or receptor function, whereas mutation of Trp(95), Trp(102), Trp(121), and Trp(214) ablated ligand binding and receptor function, and all but one of the receptors containing these mutations were not expressed at the plasma membrane. We propose that Trp(90), Trp(183), and Trp(195) are intimately involved in ligand binding, whereas Trp(95), Trp(102), Trp(121), and Trp(214) have a critical role in receptor structure or assembly.     

Interaction of active compounds from Aegle marmelos CORREA with histamine-1 receptor

The aim of this study is to determine the affinity of six active compounds of Aegle Marmelos Correa, they are (E, R)-Marmin, skimmianine, (S)-aegeline, aurapten, zeorin, and dustanin as antihistamines in histamine H1 receptor in comparison to cetirizin, diphenhydramine and chlorpheniramine as ligands comparison. Previously, in the in vitro study marmin obviously antagonized the histamine H1 receptor in a competitive manner. Methods: molecular docking to determine the interaction of ligand binding to its receptor. Lower docking score indicates more stable binding to that protein. Results: Marmin, skimmianine, aegeline, aurapten, zeorin, and dustanin were potential to develop as antihistamine agents, especially as histamine H1 receptor antagonists by interacting with amino acid residues, Asp107, Lys179, Lys191, Asn198, and Trp428 of histamine H1 receptor. Conclusions: Based on molecular docking, Amino acid residues involved in ligand protein interactions were Asp107, Lys179, Lys191, Asn198, and Trp428.     

The effect of chemical modification on the immunochemical activity of Rm-III neurotoxin from the anemone Radiantus macrodactylus]

A chemical modification was used for studying the organization of antigenic determinants of neurotoxin Rm-III from the sea anemone Radianthus macrodactylus. Immunochemical experiments were performed using competitive enzyme-linked immunosorbent assay (ELISA) with polyclonal antibodies to Rm-III. The modification affected N-terminal amino group of Gly1, Lys4, Arg13, Trp30 residues, a residue in the Lys46-Lys47-Lys48 sequence, two different residues in the Asp6-Asp7-Glu8 sequence in two samples of the toxin, and two disulphide bonds. Only the modification of the disulphide bonds led to a considerable change in the toxin's affinity to antibodies. The modification of Trp30 resulted in two-fold decrease of the toxin concentration necessary for 50% inhibition of the test-system, whereas upon modification of any other amino acid residue this concentration increased but not more than by 2.2 times. It is suggested that Rm-III sequence lacks individual residues which are of great importance for the toxin's antigenic activity, its conformation being of vital importance for the formation of the toxin's antigenic determinants.