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Binding Mechanism (binding + mechanism)
Selected AbstractsMetal-complex formation and DNA interaction of 5, 10,15,20-tetrakis(1-methyl-4-pyridiyl)-porphine: Study of the mechanistic aspectsINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 2 2010Sabriye Aydinoglu The macrocyclic porphyrin 5,10,15,20-tetrakis(1-methyl-4-pyridiyl)-porphine is studied in its ability to coordinate Cu(II) even at very low pH values and to interact, as a copper complex, with calf-thymus (CT-DNA). The kinetics and equilibria for metal-ligand complexes formation are spectrophotometrically studied, particularly focussing on the mechanistic information provided by the kinetic approach. The rate constants of complex formation is much lower than that of water exchange at Cu(II); this behavior is ascribed to an equilibrium between two porphyrin populations, only one of them being reactive. Concerning the interaction of the copper,porphyrin complex (D) with CT-DNA, it has been found that the complex binds to G,C base pairs by intercalation while forms external complex with the A,T base pairs. The kinetic results agree with a reaction mechanism that takes into account the slow shuffling from an AT-bound form (DAT) to a GC-bound form (DGC) of the copper complex (D), finally leading to a more stable DGC* intercalated form. Kinetic and equilibrium parameters for the copper complex binding to the nucleic acid are obtained, and the binding mechanism is discussed. A mechanism is proposed where D reacts simultaneously with (G,C) and (A,T) base pairs. The resulting bound forms interconvert according to a "shuffling" process, which involves formation of an intermediate (DGC) form. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 42: 79,89, 2010 [source] Oligohis-tags: mechanisms of binding to Ni2+ -NTA surfacesJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2009Steven Knecht Abstract Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni2+ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C - or N -terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni2+ -NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni2+ -NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (KD) of 14,±,1,nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd. [source] Synthetic peptides mimicking the interleukin-6/gp 130 interaction: a two-helix bundle system.JOURNAL OF PEPTIDE SCIENCE, Issue 2 2003Design, conformational studies Abstract The objective of our study was to mimic in a typical reductionist approach the molecular interactions observed at the interface between the gp 130 receptor and interleukin-6 during formation of their complex. A peptide system obtained by reproducing some of the interleukin-6/gp 130 molecular interactions into a two-helix bundle structure was investigated. The solution conformational features of this system were determined by CD and NMR techniques. The CD titration experiments demonstrated that the interaction between the designed peptides is specific and based on a well-defined stoichiometry. The NMR data confirmed some of the structural features of the binding mechanism as predicted by the rational design and indicated that under our conditions the recognition specificity and affinity can be explained by the formation of a two-helix bundle. Thus, the data reported herein represent a promising indication on how to develop new peptides able to interfere with formation of the interleukin-6/gp 130 complex. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Molecular aspects on the interaction of protoberberine, benzophenanthridine, and aristolochia group of alkaloids with nucleic acid structures and biological perspectivesMEDICINAL RESEARCH REVIEWS, Issue 5 2007Motilal Maiti Abstract Alkaloids occupy an important position in chemistry and pharmacology. Among the various alkaloids, berberine and coralyne of the protoberberine group, sanguinarine of the benzophenanthridine group, and aristololactam-,- d -glucoside of the aristolochia group have potential to form molecular complexes with nucleic acid structures and have attracted recent attention for their prospective clinical and pharmacological utility. This review highlights (i) the physicochemical properties of these alkaloids under various environmental conditions, (ii) the structure and functional aspects of various forms of deoxyribonucleic acid (DNA) (B-form, Z-form, HL -form, protonated form, and triple helical form) and ribonucleic acid (RNA) (A-form, protonated form, and triple helical form), and (iii) the interaction of these alkaloids with various polymorphic DNA and RNA structures reported by several research groups employing various analytical techniques like absorbance, fluorescence, circular dichroism, and NMR spectroscopy; electrospray ionization mass spectrometry, thermal melting, viscosity, and DNase footprinting as well as molecular modeling and thermodynamic studies to provide detailed binding mechanism at the molecular level for structure,activity relationship. Nucleic acids binding properties of these alkaloids are interpreted in relation to their biological activity. © 2006 Wiley Periodicals, Inc. Med Res Rev, 27, No. 5, 649,695, 2007 [source] Fast and automated functional classification with MED-SuMo: An application on purine-binding proteinsPROTEIN SCIENCE, Issue 4 2010Olivia Doppelt-Azeroual Abstract Ligand,protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED-SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED-SMA is an automated and fast method to classify binding sites. It is based on MED-SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora-A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED-SMA approach is demonstrated as it gathers binding sites of proteins with similar structure-activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target-based drug design and prediction of cross-reactivity and therefore potential toxic side effects. [source] Equalizer technology , Equal rights for disparate beadsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2010Eva-Maria Keidel Abstract One major limitation in proteomics is the detection and analysis of low-abundant proteins, i.e. in plasma. Several years ago, a technique to selectively enrich the relative concentration of low-abundant proteins was introduced by Boschetti and co-workers. It is based on a specific and saturable interaction of proteins to a high diversity of binding sites, realized by a hexapeptide library coupled to beads. This technology was commercialized as Equalizer beads or ProteoMiner. However, during application of ProteoMiner beads to plasma samples unexpected results questioned the proposed mode of action. Therefore, ProteoMiner beads were compared with chromatographic beads exhibiting completely different surface chemistry. Sepabeads FP-OD400 octadecyl, FP-DA400 diethylamine, FP-BU400 butyl, FP-HG400 hydroxyl and EXE056 epoxy were used. The results show that ProteoMiner or the different Sepabeads behave surprisingly similarly in the separation of complex protein mixtures. ProteoMiner beads interact with protein mixtures according to a general hydrophobic binding mechanism, where diversity in surface ligands plays only a negligible role. [source] Expression, refolding, crystallization and preliminary crystallographic study of MHC H-2Kk complexed with octapeptides and nonapeptidesACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2004Christine Kellenberger Major histocompatibility complex (MHC) molecules are heterodimeric cell-surface receptors that play a crucial role in the cellular immune response by presenting epitope peptides to T-cell antigen receptors (TCR). Although the structural basis of the peptide,MHC binding mechanism is becoming better understood, it is still difficult to predict a binding mode for an MHC of unknown structure. Therefore, as the first stage of a TCR,MHC interaction study, the crystal structures of the mouse H-2Kk molecule in complex with both an octapeptide from Influenza A virus and a nonapeptide from simian virus SV40 were solved. Here, the expression, refolding, purification and crystallization of the two complexes are reported. For the H-2Kk,HA(259,266) complex, crystals were obtained via an extensive screen using a nanodrop-dispensing robot and diffracted to 2.5,Å resolution. For the H-2Kk,SV40(560,568) complex, microscopic needles were initially obtained and their size was improved by macroseeding and a stepwise increase in precipitant concentration. Diffraction data to a resolution of 3.0,Å were collected at a synchrotron facility. [source] Preliminary X-ray crystallographic study of the receptor-binding domain of the D/C mosaic neurotoxin from Clostridium botulinumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Nipawan Nuemket Botulinum toxin (BoNT) from Clostridium botulinum OFD05, isolated from bovine botulism, is a D/C mosaic-type BoNT. BoNTs possess binding, translocation and catalytic domains. The BoNT/OFD05 binding domain exhibits significant sequence identity to BoNT/C, which requires a single ganglioside as a binding receptor on neuronal cells, while BoNT/A and BoNT/B require two receptors for specific binding. To determine the binding mechanism of BoNT/OFD05 and its ganglioside receptors on neuronal cells, recombinant BoNT/OFD05 receptor-binding domain has been expressed, purified and crystallized. Native and SeMet-derivative crystals showed X-ray diffraction to 2.8 and 3.1,Å resolution, respectively. The crystals belonged to space group P212121. [source] Potent Fluoro-oligosaccharide Probes of Adhesion in ToxoplasmosisCHEMBIOCHEM, Issue 15 2009Sarah A. Allman Abstract Unnatural, NMR- and MRI-active fluorinated sugar probes, designed and synthesised to bind to the pathogenic protein TgMIC1 from Toxoplasma gondii, were found to display binding potency equal to and above that of the natural ligand. Dissection of the binding mechanism and modes, including the first X-ray crystal structures of a fluoro-oligosaccharide bound to a lectin, demonstrate that it is possible to create effective fluorinated probe ligands for the study of, and perhaps intervention in, sugar,protein binding events. [source] Site-Specific Investigation of the Steady-State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier ProteinCHEMISTRY - A EUROPEAN JOURNAL, Issue 37 2010Dr. Clelia Cogliati Abstract The investigation of multi-site ligand,protein binding and multi-step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D 1H,15N line-shape analysis, which allows a reliable investigation of ligand binding occurring on micro- to millisecond timescales, have been extended to model a two-step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule,ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi-step events could be modelled, for several residues, with a two-step binding mechanism. The protein in the ligand-bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed. [source] Synthesis of 5,10,15,20-Tetra[4-(N -ethylpiperazinyl)phenyl]-porphyrin and Its Interaction with DNACHINESE JOURNAL OF CHEMISTRY, Issue 4 2005Guo Can-Cheng Abstract Piperazinyl-porphyrin, 5,10,15,20-tetra[4-(N -ethylpiperazinyl)phenyl]porphyrin (TEPPH2), was synthesized based on the special affinity of porphyrin to cancer cells and the antitumor activity of piperazine compounds. Its structure was characterized by UV-vis and 1H NMR spectra and elemental analysis. A model for the interaction between TEPPH2 and calf thymus DNA was built, and the binding mechanism was investigated by UV-vis and fluorescence spectra. The results indicated that TEPPH2 could intercalate into the base pairs of DNA strongly. One calf thymus DNA molecule could bind 88 TEPPH2 molecules, and the binding constant K is 8.4×106 L·mol -1. The binding number and binding constant of TEPPH2 with DNA are higher than those of the known anti-tumor drugs, tetrakis(4- N -methylpyridyl)porphine and the Schiff bases Ca/sal-his and Ni/sal-aln. [source] C-type lectin-independent interaction of complement opsonized HIV with monocyte-derived dendritic cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2005Monika Pruenster Abstract HIV directly activates the complement cascade and is, therefore, opsonized with C3-cleavage products in vivo. This cloud of C3 fragments on the viral surface may impair the interaction of the HIV envelope glycoproteins gp120/gp41 with C-type lectins expressed on immature dendritic cells (iDC). Therefore, we determined the accessibility of gp120 after opsonization and compared the interaction of DC with non-opsonized or complement-opsonized HIV. The recognition of native gp120 was drastically impaired when the virus was covered by complement. Independent of opsonization, similar amounts of HIV bound to DC. The interaction of iDC and the infection of DC-PBL co-cultures with non-opsonized virus was significantly reduced by mannan and antibodies which inhibit the ICAM-1-CR3 interaction. The binding of opsonized virus to iDC was reduced by an anti-CR3-antibody, which interferes with the binding of C3 fragments, but was not affected by mannan. Complement enhanced the HIV infection of DC and DC-PBL co-cultures significantly. Mannan did not inhibit the complement-dependent enhancement of infection. Thus, non-opsonized and opsonized HIV interacted with iDC, although the binding mechanisms seemed to differ. As HIV is opsonized in vivo, the C-type lectin-independent interaction of opsonized viruses with iDC has to be taken into account. [source] Unusual binding interactions in PDZ domain crystal structures help explain binding mechanismsPROTEIN SCIENCE, Issue 4 2010Jonathan M. Elkins Abstract PDZ domains most commonly bind the C-terminus of their protein targets. Typically the C-terminal four residues of the protein target are considered as the binding motif, particularly the C-terminal residue (P0) and third-last residue (P-2) that form the major contacts with the PDZ domain's "binding groove". We solved crystal structures of seven human PDZ domains, including five of the seven PDLIM family members. The structures of GRASP, PDLIM2, PDLIM5, and PDLIM7 show a binding mode with only the C-terminal P0 residue bound in the binding groove. Importantly, in some cases, the P-2 residue formed interactions outside of the binding groove, providing insight into the influence of residues remote from the binding groove on selectivity. In the GRASP structure, we observed both canonical and noncanonical binding in the two molecules present in the asymmetric unit making a direct comparison of these binding modes possible. In addition, structures of the PDZ domains from PDLIM1 and PDLIM4 also presented here allow comparison with canonical binding for the PDLIM PDZ domain family. Although influenced by crystal packing arrangements, the structures nevertheless show that changes in the positions of PDZ domain side-chains and the ,B helix allow noncanonical binding interactions. These interactions may be indicative of intermediate states between unbound and fully bound PDZ domain and target protein. The noncanonical "perpendicular" binding observed potentially represents the general form of a kinetic intermediate. Comparison with canonical binding suggests that the rearrangement during binding involves both the PDZ domain and its ligand. [source] Folding and binding cascades: Dynamic landscapes and population shiftsPROTEIN SCIENCE, Issue 1 2000Sandeep Kumar Abstract Whereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713,720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181,1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This extension is based on the recognition that funnels are not stationary; rather, they are dynamic, depending on the physical or binding conditions (Tsai CJ, Ma B, Nussinov R, 1999, Proc Natl Acad Sci USA 96:9970,9972). Different binding states change the surrounding environment of proteins. The changed environment is in turn expressed in shifted energy landscapes, with different shapes and distributions of populations of conformers. Hence, the function of a protein and its properties are not only decided by the static folded three-dimensional structure; they are determined by the distribution of its conformational substates, and in particular, by the redistributions of the populations under different environments. That is, protein function derives from its dynamic energy landscape, caused by changes in its surroundings. [source] Model peptide-based system used for the investigation of metal ions binding to histidine-containing polypeptidesBIOPOLYMERS, Issue 6 2010Manuela Murariu Abstract The reaction of histidine-containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI,MS) and circular dichroism (CD). The analysis of CD-spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal-bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93:497,508, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] HCV infective virions can be carried by human plateletsCELL BIOCHEMISTRY AND FUNCTION, Issue 6 2004A. Pugliese Abstract It has been previously demonstrated that platelets (PLTs) can bind and transport HIV-1 infectious virions. Hepatitis C virus (HCV),HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution towards chronic active hepatitis and hepatocellular carcinoma of HCV-related hepatitis. In the present study we investigated whether or not PLTs can carry HCV, and studied the binding mechanisms. Purified PLTs, obtained from healthy donors, HCV negative and HIV negative, were adsorbed with HCV-containing serum and then employed to infect a THP-1 monocytoid cell line. Replication of HCV was observed as shown by positivity for the E2 antigen within THP-1 cells, by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells at day 7 post-incubation with HCV-adsorbed PLTs. The binding of HCV to PLTs seems to involve fibronectin (FN), as already shown in the case of HIV-1. Indeed, treatment with RGD (Gly-Arg-Gly-Asp-Ser), the key oligopeptide of FN binding, inhibits the ability of HCV to be carried by PLTs in infective forms; the same phenomenon occurs with Mabs to FN. Moreover the infection of THP-1 cells seems to increase FN surface expression, as demonstrated by immunofluorescence tests. Copyright © 2004 John Wiley & Sons, Ltd. 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