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Conformational Changes (conformational + change)
Kinds of Conformational Changes Selected AbstractsSTUDY OF CONFORMATIONAL CHANGES OF EWE'S HOLO (NATIVE) AND APO-,-LACTALBUMIN BY SPECTROSCOPY AND TRYPSINOLYSISJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2006JEAN-MARC CHOBERT ABSTRACT Conformational changes of ewe's ,-lactalbumin (ALA) upon removal of Ca2+ were determined by surface hydrophobicity, calorimetry and circular dichroism. Native ewe's ALA resisted trypsinolysis, showing 4% maximum degradation after 20 h of hydrolysis. Removal of bound calcium by addition of either ethylenediaminetetraacetic acid or ethylene glycol bis ,-aminoethyl ether-N,N,N,N-tetraacetic acid induced major protein conformational changes, enhancing its susceptibility to trypsinolysis, and leading to complete degradation of the protein. Reversed-phase high-performance liquid chromatography profiles of tryptic hydrolysate of Ca2+ -free ALA were nearly the same through the whole enzymatic incubation period (24 h) showing the absence of sequential hydrolytic mechanism. They were characterized by the presence of five main peaks representing hydrophobic large-sized peptides. Cleaving the S-S bonds in the resulting hydrolysates with 2-mercaptoethanol gave rise to new peaks representing more hydrophilic and hydrophobic peptides. [source] Quartz Crystal Microbalance Studies on Conformational Change of Polymer Chains at InterfaceMACROMOLECULAR RAPID COMMUNICATIONS, Issue 4-5 2009Guangzhao Zhang Abstract The conformation of polymers at interface profoundly influences the interfacial properties. Quartz crystal microbalance with dissipation (QCM-D) is a newly developed technique to detect polymer behavior at interface in real time. In this article, we mainly review our QCM-D studies. Our focus is on temperature induced collapse and swelling of tethered polymer chains, pancake-to-brush transition and mushroom-to-brush of polymer chains. [source] Vibrational Spectroscopic Studies on the Disulfide Formation and Secondary Conformational Changes of Captopril,HSA Mixture after UV-B IrradiationPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2005Mei-Jane Li ABSTRACT The effects of pH and ultraviolet-B (UV-B) irradiation on the secondary structure of human serum albumin (HSA) in the absence or presence of captopril were investigated by an attenuated total reflection (ATR)/Fourier transform infrared (FTIR) spectroscopy. The UV-B exposure affecting the stability of captopril before and after captopril,HSA interaction was also examined by using confocal Raman microspectroscopy. The results indicate that the transparent pale-yellow solution for captopril,HSA mixture in all pH buffer solutions, except pH 5.0,7.0, changed into a viscous form then a gel form with UV-B exposure time. The secondary structural transformation of HSA in the captopril,HSA mixture with or without UV-B irradiation was found to shift the maxima amide I peak in IR spectra from 1652 cm,1 assigned to ,-helix structure to 1622 cm,1 because of a ,-sheet structure, which was more evident in pH 3.0, 8.0 or 9.0 buffer solutions. The Raman shift from 1653 cm,1 (,-helix) to 1670 cm,1 (,-sheet) also confirmed this result. Captopril dissolved in distilled water with or without UV-B irradiation was determined to form a captopril disulfide observed from the Raman spectra of 512 cm,1, which was exacerbated by UV-B irradiation. There was little disulfide formation in the captopril,HSA mixture even with long-term UV-B exposure, but captopril might interact with HSA to change the protein secondary structure of HSA whether there was UV-B irradiation or not. The pH of the buffer solution and captopril,HSA interaction may play more important roles in transforming the secondary structure of HSA from ,-helix to ,-sheet in the corresponding captopril,HSA mixture than UV-B exposure. The present study also implies that HSA has the capability to protect the instability of captopril in the course of UV-B irradiation. In addition, a partial unfolding of HSA induced by pH or captopril-HSA interaction under UV-B exposure is proposed. [source] Proteus in the World of Proteins: Conformational Changes in Protein KinasesARCHIV DER PHARMAZIE, Issue 4 2010Matthias Rabiller Abstract The 512 protein kinases encoded by the human genome are a prime example of nature's ability to create diversity by introducing variations to a highly conserved theme. The activity of each kinase domain is controlled by layers of regulatory mechanisms involving different combinations of post-translational modifications, intramolecular contacts, and intermolecular interactions. Ultimately, they all achieve their effect by favoring particular conformations that promote or prevent the kinase domain from catalyzing protein phosphorylation. The central role of kinases in various diseases has encouraged extensive investigations of their biological function and three-dimensional structures, yielding a more detailed understanding of the mechanisms that regulate protein kinase activity by conformational changes. In the present review, we discuss these regulatory mechanisms and show how conformational changes can be exploited for the design of specific inhibitors that lock protein kinases in inactive conformations. In addition, we highlight recent developments to monitor ligand-induced structural changes in protein kinases and for screening and identifying inhibitors that stabilize enzymatically incompetent kinase conformations. [source] Conformational change of the AcrR regulator reveals a possible mechanism of inductionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2008Ruoyu Gu The Escherichia coli AcrR multidrug-binding protein represses transcription of acrAB and is induced by many structurally unrelated cytotoxic compounds. The crystal structure of AcrR in space group P2221 has been reported previously. This P2221 structure has provided direct information about the multidrug-binding site and important residues for drug recognition. Here, a crystal structure of this regulator in space group P31 is presented. Comparison of the two AcrR structures reveals possible mechanisms of ligand binding and AcrR regulation. [source] Interaction between catalytically inactive calpain and calpastatinFEBS JOURNAL, Issue 8 2006Evidence for its occurrence in stimulated cells Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca2+, substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca2+] or extensive diffusion of calpastatin into the cytosol without affecting Ca2+ homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain,calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action. [source] Conformational changes of ,-lactoglobulin in sodium bis(2-ethylhexyl) sulfosuccinate reverse micellesFEBS JOURNAL, Issue 4 2004A fluorescence, CD study The effect of ,-lactoglobulin encapsulation in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles on the environment of protein and on Trp was analysed at different water contents (,0). CD data underlined the distortion of the ,-sheet and a less constrained tertiary structure as the ,0 increased, in agreement with a concomitant red shift and a decrease in the signal intensity obtained in steady-state fluorescence measurements. Fluorescence lifetimes, evaluated by biexponential analysis, were ,1 = 1.28 ns and ,2 = 3.36 ns in neutral water. In reverse micelles, decay-associated spectra indicated the occurrence of important environmental changes associated with ,0. Bimolecular fluorescence quenching by CCl4 and acrylamide was employed to analyse alterations in the accessibility of the two Trp residues in ,-lactoglobulin, induced by changes in ,0. The average bimolecular quenching constant <> was found not to depend on ,0, confirming the insolubility of this quencher in the aqueous interface, while <> increases with ,0. The drastic decrease with ,0 of kq, associated with the longest lifetime, , comparatively to the increase of , emphasizes the location of ,-lactoglobulin in the aqueous interfacial region especially at ,0,,10. The fact that (,0 = 30) , (water) also confirms the important conformational changes of encapsulated ,-lactoglobulin. [source] Tuning and Enhancing Photoluminescence of Light-Emitting Polymer Nanotubes through Electron-Beam IrradiationADVANCED FUNCTIONAL MATERIALS, Issue 4 2009Young Ki Hong A new method for the tuning and enhancing photoluminescence (PL) characteristics of light emitting poly (3-methylthiopnehe) (P3MT) nanotubes through E-beam irradiation under atmospheric environments is reported. An E-beam generated from a linear electron accelerator (1 MeV, 1.6,×,1013,8.0,×,1016 electrons cm,2) is irradiated onto P3MT nanotubes including an Al2O3 template. From laser confocal microscope (LCM) PL experiments, significant enhancements in the PL intensity,up to about 90 times of an isolated single strand of the E-beam irradiated P3MT nanotubes,are observed. The luminescent color of the P3MT nanotubes changes from green to red color depending on the variation of E-beam dosage. These results might originate from the de-doping effect and the conformational modification through E-beam irradiations. Conformational changes of the E-beam irradiated P3MT nanotubes are confirmed by LCM single Raman and ultraviolet-visible (UV/Vis) absorption spectra. From UV/Vis absorption spectra, it is observed that the ,,,* transition peak and the doping induced bipolaron peaks of the P3MT nanotubes dramatically vary with E-beam irradiating conditions. [source] STUDY OF CONFORMATIONAL CHANGES OF EWE'S HOLO (NATIVE) AND APO-,-LACTALBUMIN BY SPECTROSCOPY AND TRYPSINOLYSISJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2006JEAN-MARC CHOBERT ABSTRACT Conformational changes of ewe's ,-lactalbumin (ALA) upon removal of Ca2+ were determined by surface hydrophobicity, calorimetry and circular dichroism. Native ewe's ALA resisted trypsinolysis, showing 4% maximum degradation after 20 h of hydrolysis. Removal of bound calcium by addition of either ethylenediaminetetraacetic acid or ethylene glycol bis ,-aminoethyl ether-N,N,N,N-tetraacetic acid induced major protein conformational changes, enhancing its susceptibility to trypsinolysis, and leading to complete degradation of the protein. Reversed-phase high-performance liquid chromatography profiles of tryptic hydrolysate of Ca2+ -free ALA were nearly the same through the whole enzymatic incubation period (24 h) showing the absence of sequential hydrolytic mechanism. They were characterized by the presence of five main peaks representing hydrophobic large-sized peptides. Cleaving the S-S bonds in the resulting hydrolysates with 2-mercaptoethanol gave rise to new peaks representing more hydrophilic and hydrophobic peptides. [source] 1H and 13C NMR study of a series of C-9-substituted 19-norsteroidsJOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 1 2002Laurence Dallery Abstract The 1H and 13C NMR spectra of a series of 9-substituted 19-norsteroids were completely assigned using a series of 2D NMR experiments, which included 1H,1H COSY, NOESY and 1H,13C heteronuclear HETCOR and HMQC. For second-order spin systems, chemical shifts and coupling constants were obtained by simulation of the experimental spectrum. Criteria were deduced to characterize the stereochemistry of these different compounds (multiplet pattern of H-8, and variation of chemical shifts). The results allow the easy determination of the configuration at C-9. Conformational changes resulting from the substitution were studied by NMR and molecular modeling calculations (AM1). Copyright © 2001 John Wiley & Sons, Ltd. [source] Effect of thermal aging on the crystal structural characteristics of poly(tetra fluoro ethylene)POLYMER ENGINEERING & SCIENCE, Issue 11 2007Anjana Jain The residual effects of cumulative thermal aging on the crystal structural characteristics of the fluoro carbon poly(tetra fluoro ethylene) (PTFE) have been studied by X-ray diffraction methods. The initial hexagonal arrangement of the PTFE chains in a 157 helical conformation is left unaffected by the exposures to temperatures (T), up to and beyond its melting point, Tm. The unit cell registers a residual anisotropic volume expansion. The anisotropy arises from the enhanced enlargement of the basal plane dimension a compared with the axial dimension c. Conformational changes contributing to the observed increase in the chain length have been examined. Enhancement of residual crystallinity of samples aged at T's < Tm suggests that the selective thermal aging could be used as an effective tool to improve the initial crystallinity of commercially available PTFE. The activation energy for 50% enhancement in initial crystallinity has been estimated as 53.9 kJ mol,1. Aging at 400°C, a temperature above Tm, is accompanied by markedly different features viz., deterioration in crystallinity and other structural characteristics. The overall behavior of thermally aged PTFE bears a marked similarity to many polyamides. POLYM. ENG. SCI., 47:1724,1729, 2007. © 2007 Society of Plastics Engineers [source] Induced-fit or preexisting equilibrium dynamics?PROTEIN SCIENCE, Issue 4 2008Lessons from protein crystallography, MD simulations on acetylcholinesterase, implications for structure-based drug design Abstract Crystal structures of acetylcholinesterase complexed with ligands are compared with side-chain conformations accessed by native acetylcholinesterase in molecular dynamics (MD) simulations. Several crystallographic conformations of a key residue in a specific binding site are accessed in a simulation of native acetylcholinesterase, although not seen in rotomer plots. Conformational changes upon ligand binding thus involve preexisting equilibrium dynamics. Consequently, rational drug design could benefit significantly from conformations monitored by MD simulations of native targets. [source] Conformational changes induced by a single amino acid substitution in the trans -membrane domain of Vpu: Implications for HIV-1 susceptibility to channel blocking drugsPROTEIN SCIENCE, Issue 10 2007Sang Ho Park Abstract The channel-forming trans -membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight ,-helix spanning residues 6,27 with an average tilt angle of 41° in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11° compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers. [source] Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosisPROTEIN SCIENCE, Issue 8 2007Nicole A. Kruh Abstract The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD+ (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein,protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer,dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present. [source] Conformational changes in ,-endorphin as studied by electrospray ionization mass spectrometry,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2001Hui Lin Because of a wide range of physiological functions, the structure of ,-endorphin (BE) is of great interest. In this study, conformational changes in BE induced by methanol are explored with electrospray ionization-mass spectrometry (ESI-MS). Differences in the charge-state distribution (CSD) and the extent of hydrogen/deuterium (H/D) exchange were used to monitor the conformational changes. The latter experiments were conducted via time-resolved ESI-MS in a continuous-flow apparatus. Both these techniques demonstrate that BE exists in a random coil open structure in aqueous media, but it acquires a more compact conformation with increased concentration of methanol. The H/D exchange experiments reveal that BE forms 61% ,-helix in mixed solvents. Copyright © 2001 John Wiley & Sons, Ltd. [source] Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5,Å resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sitesACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2001Andrzej Wojtczak The first observation of the unique environment for thyroxine (T4) binding in tetrameric rat transthyretin (rTTR) is reported as determined by X-ray diffraction. These data revealed different modes of hormone binding in the two unique hormone-binding sites in the rat TTR tetramer channel. Differences in the orientation of thyroxine and the position of water molecules in the two binding sites further suggest a mechanism for the docking pathway of the hormone into the channel of TTR. Crystals of the rat transthyretin,thyroxine complex are isomorphous with those reported for apo rTTR and crystallized in the tetragonal space group P43212 with four independent TTR monomeric subunits in the asymmetric part of the crystal lattice. Data were collected to 2.5,Å resolution and the structure was refined to R = 20.9% for 15,384 data in the resolution range 12,2.5,Å. Similar to human TTR, the rat protein is also a 54,000,Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which differ between the human and rat sequences, none are in the thyroxine-binding domains. Analysis of these structural data reveals that the tertiary structure is similar to that of hTTR, with only small differences in the flexible loop regions on the surface of the structure. Conformational changes of the amino acids in the channel result in a hydrogen-bonded network that connects the two binding domains, in contrast to the hydrogen bonds formed along the tetramer interface in the apo transthyretin structure. These changes suggest a mechanism for the signal transmission between thyroxine-binding domains. [source] Conformational changes of enzymes adsorbed at liquid, solid interface: Relevance to enzymatic activityBIOPOLYMERS, Issue 4-5 2002S. Noinville Abstract FTIR with attenuated total reflectance spectroscopy was used to study in situ adsorption of enzymes at water,solid interfaces to better understand how conformational changes may monitor enzymatic activity. Because the adsorption process depends on hydrophobic and electrostatic interactions, conformational changes were studied as a function of the nature of the adsorbing substrates, which are hydrophobic or hydrophilic in character. The adsorption kinetics of two examples of serine enzymes, ,-chymotrypsin (,-chym) and Humicola lanuginosa lipase (HLL), were studied. The secondary structure and solvation of the adsorbed enzymes were both compared to the dissolved enzymes. The positively charged ,- chym was adsorbed on a negatively charged hydrophilic support with minor structural changes, but the negatively charged lipase had no affinity for a similar support. Both enzymes were strongly retained on the hydrophobic support. The secondary and tertiary structures of the ,-chym adsorbed on the hydrophobic support were strongly altered, which correlates to the inhibition of enzymatic hydrolysis. The specific solvation obtained for the adsorbed HLL is consistent with the existence of the open conformer in relation to the enhanced enzymatic activity at the water,hydrophobic interface. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 323,326, 2002 [source] Changes in protein conformation and dynamics upon complex formation of brain-derived neurotrophic factor and its receptor: Investigation by isotope-edited Fourier transform IR spectroscopyBIOPOLYMERS, Issue 1 2002Tiansheng Li Abstract The interactions of brain-derived neurotrophic factor (BDNF) with the extracellular domain of its receptor (trkB) are investigated by employing isotope-edited Fourier transform IR (FTIR) spectroscopy. The protein secondary structures of individual BDNF and trkB in solutions are compared with those in their complex. The temperature dependence of the secondary structures of BDNF, trkB, and their complex is also investigated. Consistent with the crystal structure, we observe by FTIR spectroscopy that BDNF in solution contains predominantly , strands (,53%) and relatively low contents of other secondary structures including , turns (,16%), disordered structures (,12%), and loops (,18%) and is deficient in , helix. We also observe that trkB in solution contains mostly , strands (52%) and little , helix. Conformational changes in both BDNF and trkB are observed upon complex formation. Specifically, upon binding of BDNF, the conformational changes in trkB appear to involve mostly , turns and disordered structures while the majority of the ,-strand conformation remains unchanged. The IR data indicate that some of the disordered structures in the loop regions are likely converted to , strands upon complex formation. The FTIR spectral data of BDNF, trkB, and their complex indicate that more amide NH groups of trkB undergo H,D exchange within the complex than those of the ligand-free receptor and that the thermal stability of trkB is decreased slightly upon binding of BDNF. The FT-Raman spectra of BDNF, trkB, and their complex show that the six intramolecular disulfide bonds of trkB undergo significant conformational changes upon binding of BDNF as a result of changes in the tertiary structure of trkB. Taken together, the FTIR and Raman data are consistent with the loosening of the tertiary structure of trkB upon binding of BDNF, which leads to more solvent exposure of the amide NH group and decreased thermal stability of trkB. This finding reveals an intriguing structural property of the neurotrophin ligand,receptor complex that is in contrast to other ligand,receptor complexes such as a cytokine,receptor complex that usually shows protection of the amide NH group and increased thermal stability upon complex formation. © 2002 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 67: 10,19, 2002; DOI 10.1002/bip.10038 [source] In-Cell NMR SpectroscopyCHEMBIOCHEM, Issue 9 2005Sina Reckel Keep it natural. NMR spectroscopy is a noninvasive spectroscopic technique that allows researchers to characterize the conformation and dynamics of proteins in their natural environment. Conformational changes, post-translational modifications or interaction with drugs can thus be studied inside living cells (see graphic). [source] Severing of F-actin by yeast cofilin is pH-independentCYTOSKELETON, Issue 9 2006Dmitry Pavlov Abstract Cofilin plays an important role in actin turnover in cells by severing actin filaments and accelerating their depolymerization. The role of pH in the severing by cofilin was examined using fluorescence microscopy. To facilitate the imaging of actin filaments and to avoid the use of rhodamine phalloidin, which competes with cofilin, ,-actin was labeled with tetramethylrhodamine cadaverine (TRC) at Gln41. The TRC-labeling inhibited actin treadmilling strongly, as measured by ,ATP release. Cofilin binding, detected via an increase in light scattering, and the subsequent conformational change in filament structure, as detected by TRC fluorescence decay, occurred 2,3 times faster at pH 6.8 than at pH 8.0. In contrast, actin filaments severing by cofilin was pH-independent. The pH-independent severing by cofilin was confirmed using actin labeled at Cys374 with Oregon Green® 488 maleimide. The depolymerization of actin by cofilin was faster at high pH. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Dynamics of P2X7 receptor pore dilation: Pharmacological and functional consequencesDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001I.P. Chessell Abstract The biophysical and functional properties of the human P2X7 receptor, expressed recombinantly in HEK-293 cells or natively in THP-1 pro-monocytic cells, were investigated in the context of pore dilation and externalisation of mature interleukin 1, (IL1,). In HEK-293 cells, the agonist 2,- and 3,-O-(4-benzoylbenzoyl)-ATP (BzATP) caused concentration-dependent inward currents (EC50 59 ,M) and with prolonged application this agonist caused a gradual increase in inward current culminating in a plateau. This increase in current was associated with pore dilation, determined by intracellular accumulation of YO-PRO-1. BzATP displayed increased potency at the pore-dilated form of the P2X7 receptor (EC50 17 ,M), and positive correlations between apparent receptor density and speed of pore dilation were observed. A monoclonal antibody selectively blocked current mediated by the naïve receptor, while currents through pore-dilated receptors were not significantly affected, which together suggest a conformational change at the level of the receptor during the dilation event. The release of mature IL1, from THP-1 cells was independent of P2X7 -mediated cell lysis, as determined by study of lactate dehydrogenase release. Moreover, using conditions designed to minimise pore dilation (using buffers containing 2 mM Ca2+ and 1 mM Mg2+), BzATP caused significant release of IL1,, but without concomitant YO-PRO-1 accumulation, indicating pore dilation is not required for IL1, release. In addition, short (4-min) incubation of THP-1 cells with BzATP (terminated by enzymatic degradation of BzATP using apyrase) resulted in significant quantities of IL1, release some 60 min later, suggesting commitment of cells to release IL1, can be triggered with only brief receptor ligation. These findings suggest that receptor expression and ligation time are critical factors for selecting multiple functional states of P2X7. Drug Dev. Res. 53:60,65, 2001. © 2001 Wiley-Liss, Inc. [source] Structured Nucleic Acid Probes for Electrochemical DevicesELECTROANALYSIS, Issue 19 2009Rebeca Miranda-Castro Abstract The use of nucleic acid with a specific sequence and a highly ordered secondary structure such as hairpins, quadruplexes and pseudoknots as biological recognition elements and switches in biosensors is rapidly increasing because of their improved features (e.g. selectivity) when compared with the traditional linear probes. Owing to the novelty, a critical outlook of their characteristics and a compilation of the latest advances are lacking. This article describes the potential of those nucleic acids probes whose molecular recognition ability relies on a conformational change (e.g. folding/unfolding mechanism) in electrochemical sensing. It provides an overview of the toolbox of assays using these probes for genosensors and aptasensors, highlighting its performance characteristics and the prospects and challenges for biosensor design. [source] Metal Ion Complementarity: Effect of Ring-Size Variation on the Conformation and Stability of Lead(II) and Cadmium(II) Complexes with Pendant-Armed CrownsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 15 2007Martín Regueiro-Figueroa Abstract The binding tendencies of the pendant-armed crown ethers L1,L3 [L1 = N,N, -bis(benzimidazol-2-ylmethyl)-1,7-diaza-12-crown-4, L2 = N,N, -bis(benzimidazol-2-ylmethyl)-1,10-diaza-15-crown-5) and L3 = N,N, -bis(benzimidazol-2-ylmethyl)-4,13-diaza-18-crown-6] towards PbII and CdII have been investigated. The X-ray crystal structure of [Cd(L3)](ClO4)2·EtOH shows that, in the solid state, the CdII ion is eight-coordinate and fits quite well into the crown hole, favouring an anti arrangement of the organic receptor. NMR measurements recorded in acetonitrile solution indicate that increasing the crown size induces a conformational change in the series of CdII complexes. The conformation goes from a syn arrangement for L1 to an anti arrangement for L3, passing through a syn [lrarr2] anti equilibrium in the complex derived from L2. On the contrary, no conformational change was observed for the corresponding PbII complexes, which have a syn conformation in all cases. These results have been confirmed by means of density functional theory (DFT) calculations performed by using the B3LYP model. The binding constants obtained from UV/Vis titration experiments in DMSO solution demonstrate that a decrease in the crown size provokes a 102 -fold enhancement of the stability for this series of CdII complexes, whereas for PbII a gradual decrease of the binding constants is observed. Receptor L1 shows a certain degree of selectivity for CdII over PbII, with a selectivity factor > 102. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Functional role of the linker region in purified human P-glycoproteinFEBS JOURNAL, Issue 13 2009Tomomi Sato Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells. P-gp possesses a ,linker region' of , 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, size-exclusion chromatography analysis and a thermo-stability assay, cleavage of the P-gp linker did not directly affect the preservation of the overall structure or the catalytic process in ATP hydrolysis. However, linker cleavage increased the kcat values both with substrate (ksub) and without substrate (kbasal), but decreased the ksub/kbasal values of all 10 tested substrates. The former result indicates that cleaving the linker activates P-gp, while the latter result suggests that the linker region maintains the tightness of coupling between the ATP hydrolase reaction and substrate recognition. Inspection of structures of the P-gp homolog, MsbA, suggests that linker-cleaved P-gp has increased ATP hydrolase activity because the linker interferes with a conformational change that accompanies the ATP hydrolase reaction. Moreover, linker cleavage affected the specificity constants [ksub/Km(D)] for some substrates (i.e. linker cleavage probably shifts the substrate specificity profile of P-gp). Thus, this result also suggests that the linker region regulates the inherent substrate specificity of P-gp. [source] Hydrolysis of acetylthiocoline, o -nitroacetanilide and o- nitrotrifluoroacetanilide by fetal bovine serum acetylcholinesteraseFEBS JOURNAL, Issue 7 2009María F. Montenegro Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze o -nitroacetanilides through aryl acylamidase activity. We have reported that BuChE tetramers and monomers of human blood plasma differ in o -nitroacetanilide (ONA) hydrolysis. The homology in quaternary structure and folding of subunits in the prevalent BuChE species () of human plasma and AChE forms of fetal bovine serum prompted us to study the esterase and amidase activities of fetal bovine serum AChE. The kcat/Km values for acetylthiocholine (ATCh), ONA and its trifluoro derivative N -(2-nitrophenyl)-trifluoroacetamide (F-ONA) were 398 × 106 m,1·min,1, 0.8 × 106 m,1·min,1, and 17.5 × 106 m,1·min,1, respectively. The lack of inhibition of amidase activity at high F-ONA concentrations makes it unlikely that there is a role for the peripheral anionic site (PAS) in F-ONA degradation, but the inhibition of ATCh, ONA and F-ONA hydrolysis by the PAS ligand fasciculin-2 points to the transit of o -nitroacetalinides near the PAS on their way to the active site. Sedimentation analysis confirmed substrate hydrolysis by tetrameric 10.9S AChE. As compared with esterase activity, amidase activity was less sensitive to guanidine hydrochloride. This reagent led to the formation of 9.3S tetramers with partially unfolded subunits. Their capacity to hydrolyze ATCh and F-ONA revealed that, despite the conformational change, the active site architecture and functionality of AChE were partially retained. [source] Effect of the disease-causing mutations identified in human ribonuclease (RNase) H2 on the activities and stabilities of yeast RNase H2 and archaeal RNase HIIFEBS JOURNAL, Issue 19 2008Muhammad S. Rohman Eukaryotic ribonuclease (RNase) H2 consists of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi,Goutières syndrome. To examine whether these mutations affect the complex stability and activity of RNase H2, three mutant proteins of His-tagged Saccharomyces cerevisiae RNase H2 (Sc-RNase H2*) were constructed. Sc-G42S*, Sc-L52R*, and Sc-K46W* contain single mutations in Sc-Rnh2Ap*, Sc-Rnh2Bp*, and Sc-Rnh2Cp*, respectively. The genes encoding the three subunits were coexpressed in Escherichia coli, and Sc-RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc-G42S* was greatly reduced compared to that of the wild-type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII. To analyze the role of this residue, four mutant proteins, Tk-G10S, Tk-G10A, Tk-G10L, and Tk-G10P, were constructed. All mutant proteins were less stable than the wild-type protein by 2.9,7.6 °C in Tm. A comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggests that the introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable. [source] In vitro expansion of DNA triplet repeats with bulge binders and different DNA polymerasesFEBS JOURNAL, Issue 18 2008Di Ouyang The expansion of DNA repeat sequences is associated with many genetic diseases in humans. Simple bulge DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. To probe the possible role of bulged structures in DNA slippage, we designed and synthesized a pair of simple chiral spirocyclic compounds [Xi Z, Ouyang D & Mu HT (2006) Bioorg Med Chem Lett16, 1180,1184], DDI-1A and DDI-1B, which mimic the molecular architecture of the enediyne antitumor antibiotic neocarzinostatin chromophore. Both compounds strongly stimulated slippage in various DNA repeats in vitro. Enhanced slippage synthesis was found to be synchronous for primer and template. CD spectra and UV thermal stability studies supported the idea that DDI-1A and DDI-1B exhibited selective binding to the DNA bulge and induced a significant conformational change in bulge DNA. The proposed mechanism for the observed in vitro expansion of long DNA is discussed. [source] Arginine-induced conformational change in the c -ring/a -subunit interface of ATP synthaseFEBS JOURNAL, Issue 9 2008Thomas Vorburger The rotational mechanism of ATP synthases requires a unique interface between the stator a subunit and the rotating c -ring to accommodate stability and smooth rotation simultaneously. The recently published c -ring crystal structure of the ATP synthase of Ilyobacter tartaricus represents the conformation in the absence of subunit a. However, in order to understand the dynamic structural processes during ion translocation, studies in the presence of subunit a are required. Here, by intersubunit Cys,Cys cross-linking, the relative topography of the interacting helical faces of subunits a and c from the I. tartaricus ATP synthase has been mapped. According to these data, the essential stator arginine (aR226) is located between the c -ring binding pocket and the cytoplasm. Furthermore, the spatially vicinal residues cT67C and cG68C in the isolated c -ring structure yielded largely asymmetric cross-linking products with aN230C of subunit a, suggesting a small, but significant conformational change of binding-site residues upon contact with subunit a. The conformational change was dependent on the positive charge of the stator arginine or the aR226H substitution. Energy-minimization calculations revealed possible modes for the interaction between the stator arginine and the c -ring. These biochemical results and structural restraints support a model in which the stator arginine operates as a pendulum, moving in and out of the binding pocket as the c -ring rotates along the interface with subunit a. This mechanism allows efficient interaction between subunit a and the c- ring and simultaneously allows almost frictionless movement against each other. [source] Phosphorylation modulates the local conformation and self-aggregation ability of a peptide from the fourth tau microtubule-binding repeatFEBS JOURNAL, Issue 19 2007Jin-Tang Du Phosphorylation of tau protein modulates both its physiological role and its aggregation into paired helical fragments, as observed in Alzheimer's diseased neurons. It is of fundamental importance to study paired helical fragment formation and its modulation by phosphorylation. This study focused on the fourth microtubule-binding repeat of tau, encompassing an abnormal phosphorylation site, Ser356. The aggregation propensities of this repeat peptide and its corresponding phosphorylated form were investigated using turbidity, thioflavin T fluorescence and electron microscopy. There is evidence for a conformational change in the fourth microtubule-binding repeat of tau peptide upon phosphorylation, as well as changes in aggregation activity. Although both tau peptides have the ability to aggregate, this is weaker in the phosphorylated peptide. This study reveals that both tau peptides are capable of self-aggregation and that phosphorylation at Ser356 can modulate this process. [source] Atrial natriuretic peptide-dependent photolabeling of a regulatory ATP-binding site on the natriuretic peptide receptor-AFEBS JOURNAL, Issue 21 2005Simon Joubert The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular ligand-binding domain, a transmembrane-spanning domain, a kinase homology domain (KHD) and a guanylyl cyclase domain. Because the presence of ATP or adenylylimidodiphosphate reduces atrial natriuretic peptide (ANP) binding and is required for maximal guanylyl cyclase activity, a direct interaction of ATP with the receptor KHD domain is plausible. Therefore, we investigated whether ATP interacts directly with a binding site on the receptor by analyzing the binding of a photoaffinity analog of ATP to membranes from human embryonic kidney 293 cells expressing the NPR-A receptor lacking the guanylyl cyclase moiety (,GC). We demonstrate that this receptor (NPR-A-,GC) can be directly labeled by 8-azido-3,-biotinyl-ATP and that labeling is highly increased following ANP treatment. The mutant receptor ,KC, which does not contain the KHD, is not labeled. Photoaffinity labeling of the NPR-A-,GC is reduced by 50% in the presence of 550 µm ATP, and competition curve fitting studies indicate a Hill slope of 2.2, suggestive of cooperative binding. This approach demonstrates directly that the interaction of ANP with its receptor modulates the binding of ATP to the KHD, probably through a conformational change in the KHD. In turn, this conformational change is essential for maximal activity. In addition, the ATP analog, 8-azido-adenylylimidodiphosphate, inhibits guanylyl cyclase activity but increases ANP binding to the extracellular domain. These results suggest that the KHD regulates ANP binding and guanylyl cyclase activity independently. [source] | |||||||||||||||||||||||||||||||||||||||||||