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Circular Dichroism Spectroscopy (circular + dichroism_spectroscopy)
Selected AbstractsInvestigation of Guanosine-Quartet Assemblies by Vibrational and Electronic Circular Dichroism Spectroscopy, a Novel Approach for Studying Supramolecular EntitiesCHEMISTRY - A EUROPEAN JOURNAL, Issue 34 2006Vladimír Setni, ka Dr. Abstract The self-assembly of guanosine-5,-hydrazide G-1 in D2O, in the presence and absence of sodium cations, has been investigated by chiroptical techniques: electronic (ECD) and the newly introduced vibrational (VCD) circular dichroism spectroscopy. Using a combination of ECD and VCD with other methods such as IR, electron microscopy, and electrospray ionization mass spectrometry (ESI-MS) it was found that G-1 produces long-range chiral aggregates consisting of G-quartets, (G-1)4, subsequently stacked into columns, [(G-1)4]n, induced by binding of metal cations between the (G-1)4 species. This process, accompanied by gelation of the sample, is highly efficient in the presence of an excess of sodium cations, leading to aggregates with strong quartet,quartet interaction. Thermally induced conformational changes and conformational stability of guanosine-5,-hydrazide assemblies were studied by chiroptical techniques and the melting temperature of the hydrogels formed was obtained. The temperature-dependent experiments indicate that the long-range supramolecular aggregates are dissociated by increasing temperature into less ordered species, monomers, or other intermediates in equilibrium, as indicated by MS experiments. Proces samoskladby guanosin-5,-hydrazidu G-1 v D2O roztocích, bez a za p,ítomnosti sodných iont,, byl studován chiroptickými metodami: spektroskopií elektronového (ECD) a nov, také vibra,ního (VCD) cirkulárního dichroismu. Kombinací spektroskopie ECD a VCD s jinými metodami jako I, spektroskopií, elektronovou mikroskopií a hmotnostní spektrometrií s ionizací elektrosprejem (ESI-MS) bylo zji,t,no, ,e molekula G-1 vytvá,í rozsáhlé chirální útvary slo,ené z G-kvartet,, (G-1)4, které mohou být následn, za p,ítomnosti kationt, spojeny patrovými interakcemi za vzniku uspo,ádaných vláken, [(G-1)4]n. Tento proces spojený s gelovat,ním vzorku je vysoce ú,inný v p,ítomnosti sodných kationt,, které vedou ke vzniku agregát, fixovaných silnými interakcemi mezi jednotlivými kvartety. Teplotn, indukované konforma,ní zm,ny a konforma,ní stabilita samoskladných útvar, tvo,ených guanosin-5,-hydrazidem byly studovány chiroptickými metodami a byly získány teploty tání p,ipravených hydrogel,. Teplotn, závislé experimenty ukázaly, ,e rozsáhlé supramolekulární útvary jsou se vzr,stající teplotou rozru,ovány za vzniku útvar, mén, usp,ádaných, monomer, nebo n,kterých intermediát,, které jsou ve vzájemné rovnováze, jak bylo indikováno MS experimenty. [source] Novel Secondary Structure of Calcitonin in Solid State as Revealed by Circular Dichroism SpectroscopyCHINESE JOURNAL OF CHEMISTRY, Issue 7 2002Hai-Ning Du Abstract The solid-state circular dichroic study reveals that salmon calcitonin presents a typical ,-helical structure while human calcitonin appears to form a ,-sheet in solid state, although both of them adopt random coil structures in aqueous solution. [source] Site-directed mutagenesis of the active site serine290 in flavanone 3,-hydroxylase from Petunia hybridaFEBS JOURNAL, Issue 3 2000Richard Luka Flavanone 3,-hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In the presence of oxygen and ferrous ions the enzyme couples the oxidative decarboxylation of 2-oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols. The hydroxylase had been cloned from Petunia hybrida and expressed in Escherichia coli, and a rapid isolation method for the highly active, recombinant enzyme had been developed. Sequence alignments of the Petunia hydroxylase with various hydroxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved amino acids, including a strictly conserved serine residue (Ser290). This serine was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2-oxoglutarate-dependent enzymes. The mutant enzymes were expressed in E. coli and purified to homogeneity. The catalytic activities of [Thr290]FHT and [Ala290]FHT were still significant, albeit greatly reduced to 20 and 8%, respectively, in comparison to the wild-type enzyme, whereas the activity of [Val290]FHT was negligible (about 1%). Kinetic analyses of purified wild-type and mutant enzymes revealed the functional significance of Ser290 for 2-oxoglutarate-binding. The spatial configurations of the related Fe(II)-dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and provide the lead structures for the conformation of other dioxygenases. Circular dichroism spectroscopy was employed to compare the conformation of pure flavanone 3,-hydroxylase with that of isopenicillin N synthase. A double minimum in the far ultraviolet region at 222 nm and 208,210 nm and a maximum at 191,193 nm which are characteristic for ,-helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship. Furthermore, the spectrum remained unchanged after addition of either ferrous ions, 2-oxoglutarate or both of these cofactors, ruling out a significant conformational change of the enzyme on cofactor-binding. [source] A ,-amino acid modified heptapeptide containing a designed recognition element disrupts fibrillization of the amyloid ,-peptideJOURNAL OF PEPTIDE SCIENCE, Issue 9 2010Valeria Castelletto Abstract We study the complex formation of a peptide ,A,AKLVFF, previously developed by our group, with A,(1,42) in aqueous solution. Circular dichroism spectroscopy is used to probe the interactions between ,A,AKLVFF and A,(1,42), and to study the secondary structure of the species in solution. Thioflavin T fluorescence spectroscopy shows that the population of fibers is higher in ,A,AKLVFF/A,(1,42) mixtures compared to pure A,(1,42) solutions. TEM and cryo-TEM demonstrate that co-incubation of ,A,AKLVFF with A,(1,42) causes the formation of extended dense networks of branched fibrils, very different from the straight fibrils observed for A,(1,42) alone. Neurotoxicity assays show that although ,A,AKLVFF alters the fibrillization of A,(1,42), it does not decrease the neurotoxicity, which suggests that toxic oligomeric A,(1,42) species are still present in the ,A,AKLVFF/A,(1,42) mixtures. Our results show that our designed peptide binds to A,(1,42) and changes the amyloid fibril morphology. This is shown to not necessarily translate into reduced toxicity. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. [source] Design of a minimal protein oligomerization domain by a structural approachPROTEIN SCIENCE, Issue 12 2000Peter Burkhard Abstract Because of the simplicity and regularity of the ,-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly ,-helical and 100% dimeric under physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system. [source] Circular dichroism spectroscopy reveals invariant conformation of guanine runs in DNABIOPOLYMERS, Issue 4-5 2002Jaroslav Kypr Abstract We demonstrate that the characteristic circular dichroism (CD) features of the parallel-stranded DNA tetraplex of d(G4), especially the strong band at 260 nm, are characteristic for the B and A forms of the antiparallel duplex of d(C4G4). Hence, this band evidently originates from intrastrand guanine,guanine stacking, which is therefore very similar in the duplex and tetraplex DNA. In addition, the same type of the CD spectrum is provided by the ordered single strand of d(GA)10. This observation suggests that the ordered single strand of d(GA)10 is stabilized by a core of guanines stacked like in the parallel tetraplex. This view is used to start the modeling of the molecular structure of the ordered d(GA)10 single strand. Our studies suggest that guanine itself is strong enough to stabilize various secondary structures of DNA, which is a property relevant to thinking about the origin and evolution of molecular replicators. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 275,277, 2002 [source] Circular dichroism spectroscopy of conformers of (guanine + adenine) repeat strands of DNACHIRALITY, Issue 7 2003Iva Kejnovská Abstract (Guanine+adenine) strands of DNA are known to associate into guanine tetraplexes, homodimerize into parallel or antiparallel duplexes, and fold into a cooperatively melting single strand resembling the protein alpha helix. Using CD spectroscopy and other methods, we studied how this conformational polymorphism depended on the primary structure of DNA. The study showed that d(GGGA)5 and d(GGA)7 associated into homoduplexes at low salt or in the presence of LiCl but were prone to guanine tetraplex formation, especially in the presence of KCl. In addition, they yielded essentially the same CD spectrum in the presence of ethanol as observed with the ordered single strand of d(GA)10. Strands of d(GA)10, d(GGAA)5, d(GAA)7, and d(GAAA)5 associated into homoduplexes in both LiCl and KCl solutions, but not into guanine tetraplexes. d(GAAA)5 and d(GAA)7 further failed to form the single-stranded conformer in aqueous ethanol. Adenine protonation, however, stabilized the single-stranded conformer even in these adenine-rich fragments. The ordered single strands, homoduplexes as well as the guanine tetraplexes, all provided strikingly similar CD spectra, indicating that all of the conformers shared similar base stacking geometries. The increasing adenine content only decreased the conformer thermostability. Chirality 15:584,592, 2003. © 2003 Wiley-Liss, Inc. [source] Sequence-dependent Interactions of Cationic Naphthalimides and PolynucleotidesPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007Sun McMasters The binding interactions of three naphthalimide derivatives with heteropoly nucleic acids have been evaluated using fluorescence, absorption and circular dichroism spectroscopies. Mono- and bifunctionalized naphthalimides exhibit sequence-dependent variations in their affinity toward DNA. The heteropoly nucleic acids, [Poly(dA-dT)]2 and [Poly(dG-dC)]2, as well as calf thymus (CT) DNA, were used to understand the factors that govern binding strength and selectivity. Sequence selectivity was addressed by determining the binding constants as a function of polynucleotide composition according to the noncooperative McGhee,von Hippel binding model. Binding affinities toward [poly(dA-dT)]2 were the largest for spermine-substituted naphthalimides (Kb = 2,6 × 106 m,1). The association constants for complex formation between the cationic naphthalmides and [poly(dG-dC)]2 or CT DNA (58% A-T content) were 2,500 times smaller, depending on the naphthalmide,polynucleotide pair. The binding modes were also assessed using a combination of induced circular dichroism and salt effects to determine whether the naphthalimides associate with DNA through intercalative, electrostatic or groove-binding. The results show that the monofunctionalized spermine and pyridinium-substituted naphthalimides associate with DNA through electrostatic interactions. In contrast, intercalative interactions are predominant in the complex formed between the bifunctionalized spermine compound and all of the polynucleotides. [source] Developing a New Class of Axial Chiral Phosphorus Ligands: Preparation and Characterization of Enantiopure Atropisomeric PhosphininesCHEMISTRY - A EUROPEAN JOURNAL, Issue 16 2008Christian Müller Dr. Abstract Both enantiomers of the first atropisomeric phosphinine (1) have been isolated by using analytical HPLC on a chiral stationary phase. The enrichment of one enantiomer and a subsequent investigation into its racemization kinetics revealed a barrier for internal rotation of ,=(109.5±0.5),kJ,mol,1, which is in excellent agreement with the theoretically predicted value of ,=116,kJ,mol,1. Further analysis with UV and circular dichroism spectroscopies and density functional theory calculations led to the determination and assignment of the absolute configurations of both enantiomers. These results are the basis for future investigations into this new class of axially chiral phosphinine-based ligands and their possible applications in asymmetric homogeneous catalysis. [source] Physical characterization of plakophilin 1 reconstituted with and without zincFEBS JOURNAL, Issue 14 2000Ilse Hofmann Plakophilin 1 (PKP1) belongs to the arm -repeat protein family which is characterized by the presence of a conserved 42-amino-acid motif. Despite individual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions. PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes. In surface plasmon resonance detection experiments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium. Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The , helix content of PKP1 was considerably higher when reconstituted with zinc than without. By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild-type PKP1. The zinc-reconstituted protein formed globular particles of 21.9 ± 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s20,w) for zinc-containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 × 10,7 cm2·s,1. The molecular mass of 2.44 × 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation. [source] Supramolecular Organization of ssDNA-Templated ,-Conjugated Oligomers via Hydrogen BondingADVANCED MATERIALS, Issue 10-11 2009Mathieu Surin The templated self-assembly of water-soluble ,-conjugated molecules bearing a diaminotriazine moiety H-bonding to a single-strand oligothymine template leads to defined structures. We study these assemblies with molecular modeling, circular dichroism spectroscopy, and scanning probe microscopy, to get a better understanding of the factors governing the supramolecular organization and structural order. [source] Synthesis and enantioselectivities of soluble polymers incorporating optically active binaphthyl and binaphtholJOURNAL OF APPLIED POLYMER SCIENCE, Issue 2 2007Xiaowei Zou Abstract A polymer (P-1) was synthesized through the polymerization of (S)-6,6,-dibromo-3,3,-dibutyl-1,1,-binaphthol with (S)-2,2,-dioctoxy-1,1,-binaphthyl-6,6,-boronic acid in a Pd-catalyzed Suzuki reaction, and another polymer (P-2) was synthesized through the polymerization of (S)-6,6,-dibromo-3,3,-dibutyl-1,1,-binaphthol with (S)-6,6,-diethynyl-2,2,-dioctoxy-1,1,-binaphthyl in a Pd-catalyzed Sonogashira reaction. The two polymers showed good solubility in some common solvents and were characterized with NMR, Fourier transform infrared, gel permeation chromatography, and circular dichroism spectroscopy. The application of the chiral monomers and polymers in the asymmetric addition of diethyl zinc to benzaldehyde was studied. The results indicated that P-1, P-2, and the monomer (S)-3,3,-dibutyl-1,1,-binaphthol were efficient ligands in the asymmetric addition of diethyl zinc to benzaldehyde. The chiral polymer ligands P-1 and P-2 were more efficient than their monomeric version, (S)-3,3,-dibutyl-1,1,-binaphthol, and could be easily recovered and reused without a loss of catalytic activity or enantioselectivity. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007 [source] Potential implications of endogenous aldehydes in ,-amyloid misfolding, oligomerization and fibrillogenesisJOURNAL OF NEUROCHEMISTRY, Issue 5 2006Kun Chen Abstract Aldehydes are capable of inducing protein cross-linkage. An increase in aldehydes has been found in Alzheimer's disease. Formaldehyde and methylglyoxal are produced via deamination of, respectively, methylamine and aminoacetone catalyzed by semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6. The enzyme is located on the outer surface of the vasculature, where amyloidosis is often initiated. A high SSAO level has been identified as a risk factor for vascular disorders. Serum SSAO activity has been found to be increased in Alzheimer's patients. Malondialdehyde and 4-hydroxynonenal are derived from lipid peroxidation under oxidative stress, which is also associated with Alzheimer's disease. Aldehydes may potentially play roles in ,-amyloid aggregation related to the pathology of Alzheimer's disease. In the present study, thioflavin-T fluorometry, dynamic light scattering, circular dichroism spectroscopy and atomic force microscopy were employed to reveal the effect of endogenous aldehydes on ,-amyloid at different stages, i.e. ,-sheet formation, oligomerization and fibrillogenesis. Formaldehyde, methylglyoxal and malondialdehyde and, to a lesser extent, 4-hydroxynonenal are not only capable of enhancing the rate of formation of ,-amyloid ,-sheets, oligomers and protofibrils but also of increasing the size of the aggregates. The possible relevance to Alzheimer's disease of the effects of these aldehydes on ,-amyloid deposition is discussed. [source] Synthesis of pathological and nonpathological human exon 1 huntingtinJOURNAL OF PEPTIDE SCIENCE, Issue 7 2010David Singer Abstract Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG-triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly-Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67-amino acid-long polypeptide plus a variable poly-Q stretch, is sufficient to cause full HD-like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109-amino acid-long exon 1 huntingtin peptide including a poly-Q stretch of 42 glutamines. This microwave-assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90-amino acid long including a poly-Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha-helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large-scale pre-screenings for aggregation inhibitors, further structural analyses as well as protein,protein interaction studies. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. [source] The conformation of fusogenic B18 peptide in surfactant solutions,JOURNAL OF PEPTIDE SCIENCE, Issue 4 2008Sandra Rocha Abstract The interaction of B18 peptide with surfactants has been studied by circular dichroism spectroscopy and fluorescence measurements. B18 is the fusogenic motif of the fertilization sea urchin protein. The peptide forms an ,-helix structure when interacting with positively or negatively charged surfactants below and above the critical micellar concentration (CMC). The ,-helix formation is due to binding of surfactant monomers rather than the formation of surfactant micelles on the peptide. Fluorescence measurements show that the CMC of the negatively charged surfactant increases in the presence of B18, supporting the fact that there is a strong interaction between the peptide and monomers. Nonionic surfactant monomers have no effect on the peptide structure, whereas the micelles induce an ,-helical conformation. In this case the helix stabilization results from the formation of surfactant micelles on the peptide. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Evidence that WbpD is an N -acetyltransferase belonging to the hexapeptide acyltransferase superfamily and an important protein for O-antigen biosynthesis in Pseudomonas aeruginosa PAO1MOLECULAR MICROBIOLOGY, Issue 5 2005Cory Q. Wenzel Summary Di- N -acetylated uronic acid residues are unique sugar moieties observed in the lipopolysaccharides (LPS) of respiratory pathogens including several serotypes of Pseudomonas aeruginosa and several species of Bordetella. WbpD of P. aeruginosa PAO1 (serotype O5) is a putative 3- N -acetyltransferase that has been implicated in the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy- d -mannuronic acid [UDP- d -Man(2NAc3NAc)A], a precursor for the d -Man(2NAc3NAc)A residues in the B-band O antigen of this bacterium. A chromosomal knockout mutant of wbpD is incapable of producing either long-chain B-band O antigen (, 2 repeating units) or semi-rough LPS (lipid A-core + one repeat). Adding wbpD in trans restored LPS production to the wild-type level; this indicates that wbpD is important for biosynthesis of individual B-band O-antigen repeating units. WbpD contains left-handed beta-helical (L,H) structure as observed by Conserved Domain analysis and in silico secondary and tertiary structure predictions. This feature suggested that WbpD belongs to the hexapeptide acyltransferase (HexAT) superfamily of enzymes. WbpD was overexpressed as an N-terminally histidine-tagged fusion protein (His6,WbpD) and purified to >,95% purity. The protein was subjected to Far-UV circular dichroism spectroscopy, and the data revealed that WbpD contains left-handed helical structure, which substantiated in silico predictions made earlier. Results from SDS-PAGE, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), and gel filtration analyses indicated that His6 -WbpD has trimeric organization, consistent with the quaternary structure of HexATs. The binding of acetyl-CoA by WbpD was demonstrated by MALDI-TOF MS, suggesting that WbpD is an acetyltransferase that utilizes a direct-transfer reaction mechanism. Incubation of WbpD with acetyl-CoA significantly enhanced the stability of the protein and prevented precipitation over a course of 14 days. As a substrate for studying the enzymatic activity of WbpD is unavailable at present, a structure-based model for the L,H domain of WbpD was generated. Comparisons between this model and the L,H domains of known HexATs suggested that Lys136 plays a role in acetyl-CoA binding. A K136A site-directed mutant construct could only partially complement the wbpD knockout, and this mutation also reduced the stabilizing effects of acetyl-CoA, while a K136R mutation showed no discernible effect on complementation of the wbpD mutant or the stabilizing effects of acetyl-CoA on the purified mutant protein. A modified pathway was proposed for the biosynthesis of UDP- d -Man(2NAc3NAc)A, in which WbpD is involved in the catalysis of the fourth step by acting as a UDP-2-acetamido-3-amino-2,3-dideoxy- d -glucuronic acid 3- N -acetyltransferase. [source] The refolding of different ,-fetoprotein variantsPROTEIN SCIENCE, Issue 9 2006Susanna S.J. Leong Abstract The effect of glycosylation on AFP foldability was investigated by parallel quantitative and qualitative analyses of the refolding of glycosylated and nonglycosylated AFP variants. Both variants were successfully refolded by dialysis from the denatured-reduced state, attaining comparable "refolded peak" profiles and refolding yields as determined by reversed-phase HPLC analysis. Both refolded variants also showed comparable spectroscopic fingerprints to each other and to their native counterparts, as determined by circular dichroism spectroscopy. Inclusion body-derived AFP was also readily refolded via dilution under the same redox conditions as dialysis refolding, showing comparable circular dichroism fingerprints as native nonglycosylated AFP. Quantitative analyses of inclusion body-derived AFP showed sensitivity of AFP aggregation to proteinaceous and nonproteinaceous inclusion body contaminants, where refolding yields increased with increasing AFP purity. All of the refolded AFP variants showed positive responses in ELISA that corresponded with the attainment of a bioactive conformation. Contrary to previous reports that the denaturation of cord serum AFP is an irreversible process, these results clearly show the reversibility of AFP denaturation when refolded under a redox-controlled environment, which promotes correct oxidative disulfide shuffling. The successful refolding of inclusion body-derived AFP suggests that fatty acid binding may not be required for the attainment of a rigid AFP tertiary structure, contrary to earlier studies. The overall results from this work demonstrate that foldability of the AFP molecule from its denatured-reduced state is independent of its starting source, the presence or absence of glycosylation and fatty acids, and the refolding method used (dialysis or dilution). [source] Structure and stability of the ankyrin domain of the Drosophila Notch receptorPROTEIN SCIENCE, Issue 11 2003Mark E. Zweifel RMSD, root mean square deviation; CD, circular dichroism spectroscopy Abstract The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 Ĺ resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions. [source] Design, synthesis, and characterization of a novel hemoproteinPROTEIN SCIENCE, Issue 2 2001Zhijin Xu Abstract Here we describe a synthetic protein (6H7H) designed to bind four heme groups via bis,histidine axial ligation. The hemes are designed to bind perpendicular to another in an orientation that mimics the relative geometry of the two heme a groups in the active site of cytochrome c oxidase. Our newly developed protein-design program, called CORE, was implemented in the design of this novel hemoprotein. Heme titration studies resolved four distinct KD values (KD1 = 80 nM, KD2 = 18 nM, KD3 , 3 mM, KD4 , 570 nM, with KD3 × K D4 = 1700); positive cooperativity in binding between the first and second heme, as well as substantial positive cooperativity between the third and forth heme, was observed. Chemical and thermal denaturation studies reveal a stable protein with native-like properties. Visible circular dichroism spectroscopy of holo-6H7H indicates excitonic coupling between heme groups. Further electrochemical and spectroscopic characterization of the holo-protein support a structure that is consistent with the predefined target structure. [source] Redox regulation of cyclophilin A by glutathionylationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2006Pietro Ghezzi Abstract Using redox proteomics techniques to characterize the thiol status of proteins in human T lymphocytes, we identified cyclophilin,A (CypA) as a specifically oxidized protein early after mitogen activation. CypA is an abundantly expressed cytosolic protein, target of the immunosuppressive drug cyclosporin,A (CsA), for which a variety of functions has been described. In this study, we could identify CypA as a protein undergoing glutathionylation in vivo. Using MALDI-MS we identified Cys52 and Cys62 as targets of glutathionylation in T,lymphocytes, and, using bioinformatic tools, we defined the reasons for the susceptibility of these residues to the modification. In addition, we found by circular dichroism spectroscopy that glutathionylation has an important impact on the secondary structure of CypA. Finally, we suggest that glutathionylation of CypA may have biological implications and that CypA may play a key role in redox regulation of immunity. [source] Ion channel activity of transmembrane segment 6 of Escherichia coli proton-dependent manganese transporterBIOPOLYMERS, Issue 8 2010uková Abstract Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary-active transporter MntH (Proton-dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure,function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi-state ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His211 were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings could contribute to understanding the structure,function relationship at the molecular level. However it remains unclear to what extent the peptide-specific channel activity represents a functional aspect of the full-length membrane carrier protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 718,726, 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] Bioencapsulation of apomyoglobin in nanoporous organosilica sol,gel glasses: Influence of the siloxane network on the conformation and stability of a model proteinBIOPOLYMERS, Issue 11 2009Bouzid Menaa Abstract Nanoporous sol,gel glasses were used as host materials for the encapsulation of apomyoglobin, a model protein employed to probe in a rational manner the important factors that influence the protein conformation and stability in silica-based materials. The transparent glasses were prepared from tetramethoxysilane (TMOS) and modified with a series of mono-, di- and tri-substituted alkoxysilanes, RnSi(OCH3)4,n (R = methyl-, n = 1; 2; 3) of different molar content (5, 10, 15%) to obtain the decrease of the siloxane linkage (SiOSi). The conformation and thermal stability of apomyoglobin characterized by circular dichroism spectroscopy (CD) was related to the structure of the silica host matrix characterized by 29Si MAS NMR and N2 adsorption. We observed that the protein transits from an unfolded state in unmodified glass (TMOS) to a native-like helical state in the organically modified glasses, but also that the secondary structure of the protein was enhanced by the decrease of the siloxane network with the methyl modification (n = 0 < n = 1 < n = 2 < n = 3; 0 < 5 < 10 < 15 mol %). In 15% trimethyl-modified glass, the protein even reached a maximum molar helicity (,24,000 deg. cm2 mol,1) comparable to the stable folded heme-bound holoprotein in solution. The protein conformation and stability induced by the change of its microlocal environment (surface hydration, crowding effects, microstructure of the host matrix) were discussed owing to this trend dependency. These results can have an important impact for the design of new efficient biomaterials (sensors or implanted devices) in which properly folded protein is necessary. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 895,906, 2009. 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] Guanine quadruplex formation by RNA/DNA hybrid analogs of Oxytricha telomere G4T4G4 fragmentBIOPOLYMERS, Issue 10 2008Jitka Vondru Abstract Using circular dichroism spectroscopy, gel electrophoresis, and ultraviolet absorption spectroscopy, we have studied quadruplex folding of RNA/DNA analogs of the Oxytricha telomere fragment, G4T4G4, which forms the well-known basket-type, antiparallel quadruplex. We have substituted riboguanines (g) for deoxyriboguanines (G) in the positions G1, G9, G4, and G12; these positions form the terminal tetrads of the G4T4G4 quadruplex and adopt syn, syn, anti, and anti glycosidic geometries, respectively. We show that substitution of a single sugar was able to change the quadruplex topology. With the exception of G4T4G3g, which adopted an antiparallel structure, all the RNA/DNA hybrid analogs formed parallel, bimolecular quadruplexes in concentrated solution at low salt. In dilute solutions (,0.1 mM nucleoside), the RNA/DNA hybrids substituted at positions 4 or 12 adopted antiparallel quadruplexes, which were especially stable in Na+ solutions. The hybrids substituted at positions 1 and 9 preferably formed parallel quadruplexes, which were more stable than the nonmodified G4T4G4 quadruplex in K+ solutions. Substitutions near the 3,end of the molecule affected folding more than substitutions near the 5,end. The ability to control quadruplex folding will allow further studies of biophysical and biological properties of the various folding topologies. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 797,806, 2008. 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] H93G myoglobin cavity mutant as versatile template for modeling heme proteins: Magnetic circular dichroism studies of thiolate- and imidazole-ligated complexesBIOPOLYMERS, Issue 4-5 2002John H. Dawson Abstract Recent ligand binding and spectroscopic investigations of the myoglobin H93G cavity mutant are reviewed, revealing it to be a versatile template for the preparation of model heme complexes of defined structure. The H93G myoglobin cavity mutant is shown to be capable of forming mixed ligand adducts because of the difference in accessibility of the two sides of the ferric heme iron. With imidazole bound in the proximal cavity, H93G myoglobin also forms reasonably stable oxyferrous and oxoferryl derivatives, thereby providing a potential system to use for the study of such complexes with proximal ligands other than imidazole. In addition, thiolate-ligated ferric H93G derivatives are described that serve as spectroscopic models for the high-spin ferric state of cytochrome P450. All of the complexes described are characterized with magnetic circular dichroism spectroscopy, and they are compared to the appropriate derivatives of native myoglobin and P450. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 200,206, 2002 [source] A Recombinant Glutamine-Binding Protein from Escherichia coli: Effect of Ligand-Binding on Protein Conformational DynamicsBIOTECHNOLOGY PROGRESS, Issue 6 2004Petr Herman We have investigated the effect of the binding of glutamine on the conformational dynamics of the recombinant glutamine binding protein (GlnBP) from Escherichia coli by steady-state and time-resolved fluorescence techniques. The structural stability of the protein was also studied by far-UV circular dichroism spectroscopy in the range of temperature between 25 and 80 °C. The results showed that the interaction of the protein with the ligand resulted in a marked change of the structural and conformational dynamics features of the protein. In particular, the fluorescence and circular dichroism data showed that the presence of glutamine resulted in a dramatic increase of the protein thermal stability of about 10 °C. In addition, the fluorescence time-resolved data pointed out that both in the absence and in the presence of glutamine the protein structure was highly rigid with small amplitude of segmental motion up to 65 °C and a low accessibility of the protein tryptophan residues to acrylamide. The obtained results on the structural properties of the recombinant glutamine-binding protein in the absence and in the presence of glutamine can contribute to a better understanding of the transport-related functions of the protein and structurally similar periplasmic transport proteins, as well as to the design and development of new biotechnological applications of this class of proteins. [source] Amphipathic control of the 310 -/,-helix equilibrium in synthetic peptidesCHEMICAL BIOLOGY & DRUG DESIGN, Issue 2 2001L. G. J. Hammarström Abstract: A series of short, amphipathic peptides incorporating 80% C,,C, -disubstituted glycines has been prepared to investigate amphipathicity as a helix-stabilizing effect. The peptides were designed to adopt 310 - or ,-helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1,:,1 acetonitrile/water; 9,:,1 acetonitrile/water; 9,:,1 acetonitrile/TFE; 25 mm SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 310 -helix in peptides containing amino acids with large side-chains, as with 1-aminocyclohexane-1-carboxylic acid (Ac6c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 310 -helix. [source] Studies on the conformational properties of CP-1042,55, the hinge region of CP-10, using circular dichroism and RP-HPLCCHEMICAL BIOLOGY & DRUG DESIGN, Issue 6 2000E. Lazoura Abstract: The conformational properties of CP-1042,55, a peptide corresponding to the hinge region of CP-10, were investigated using circular dichroism spectroscopy and reverse-phase high-performance liquid chromatography (RP-HPLC). The circular dichroism studies indicated that CP-1042,55 formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mm sodium dodecyl sulfate, which comprised a mixture of ,-helix and ,-sheet. The effect of temperature on the conformation of CP-1042,55 was investigated between 5 and 40°C, with very small changes in the spectra being observed.RP-HPLC was then used to investigate the effect of temperature on the conformation of CP-1042,55 in the presence of a hydrophobic surface. Using a C18 -adsorbent, CP-1042,55 exhibited a conformational transition at 25°C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near-planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25°C in theRP-HPLC system most likely corresponds to the unfolding of an ,-helical and/or ,-sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment. [source] Dynamic Supramolecular Polymers Based on Benzene-1,3,5-tricarboxamides: The Influence of Amide Connectivity on Aggregate Stability and Amplification of ChiralityCHEMISTRY - A EUROPEAN JOURNAL, Issue 3 2010Patrick Abstract N-Centred benzene-1,3,5-tricarboxamides (N-BTAs) composed of chiral and achiral alkyl substituents were synthesised and their solid-state behaviour and self-assembly in dilute alkane solutions were investigated. A combination of differential scanning calorimetry (DSC), polarisation optical microscopy (POM) and X-ray diffraction revealed that the chiral N-BTA derivatives with branched 3,7-dimethyloctanoyl chains were liquid crystalline and the mesophase was assigned as Colho. In contrast, N-BTA derivatives with linear tetradecanoyl or octanoyl chains lacked a mesophase and were obtained as crystalline compounds. Variable-temperature infrared spectroscopy showed the presence of threefold, intermolecular hydrogen bonding between neighbouring molecules in the mesophase of the chiral N-BTAs. In the crystalline state at room temperature a more complicated packing between the molecules was observed. Ultraviolet and circular dichroism spectroscopy on dilute solutions of N-BTAs revealed a cooperative self-assembly behaviour of the N-BTA molecules into supramolecular polymers with preferred helicity when chiral alkyl chains were present. Both the sergeants-and-soldiers as well as the majority-rules principles were operative in stacks of N-BTAs. In fact, the self-assembly of N-BTAs resembles closely that of their carbonyl (CO)-centred counterparts, with the exception that aggregation is weaker and amplification of chirality is less pronounced. The differences in the self-assembly of N- and CO-BTAs were analysed by density functional theory (DFT) calculations. These reveal a substantially lower interaction energy between the monomeric units in the supramolecular polymers of N-BTAs. The lower interaction energy is due to the higher energy penalty for rotation around the PhNH bond compared to the PhCO bond and the diminished magnitude of dipole,dipole interactions. Finally, we observed that mixed stacks are formed in dilute solution when mixing N-BTAs and CO BTAs. [source] Application of the Perimeter Model to the Assignment of the Electronic Absorption Spectra of Gold(III) Hexaphyrins with [4n+2] and [4n] ,-Electron SystemsCHEMISTRY - A EUROPEAN JOURNAL, Issue 15 2009Atsuya Muranaka Dr. Abstract Expanded porphyrins: The electronic excited states of two forms of meso -hexakis(pentafluorophenyl)-substituted gold(III) hexaphyrin(1.1.1.1.1.1), such as that depicted, have been investigated by density functional calculations and magnetic circular dichroism spectroscopy to assign their low-energy excited singlet states. The electronic excited states of two forms of meso -hexakis(pentafluorophenyl)-substituted gold(III) hexaphyrin(1.1.1.1.1.1) have been investigated by density functional calculations and magnetic circular dichroism (MCD) spectroscopy, in order to assign their low-energy excited singlet states. We found that the perimeter model can be successfully applied to the interpretation of the electronic states. In the case of the neutral forms (Au2 -N, Au-N), the absorption bands observed in the NIR and visible region can be assigned to ,,,* transitions referred to as the L and B bands, respectively, analogous to the Q and Soret bands of regular porphyrins. In marked contrast with the neutral forms, the absorption bands of the reduced forms (Au2 -R and Au-R) are attributed to ,,,* transitions involving six frontier molecular , orbitals. By applying the 4N -electron perimeter model, the six orbitals are labeled as h,, h+, s,, s+, l,, and l+, while the observed absorption bands can be assigned to the S, N1, N2, P1, and P2 transitions, in order of increasing energy. [source] Enantiospecific Syntheses of Copper Cubanes, Double-Stranded Copper/Palladium Helicates, and a (Dilithium),Dinickel Coronate from Enantiomerically Pure Bis-1,3-diketones,Solid-State Self-Organization Towards Wirelike Copper/Palladium Strands,CHEMISTRY - A EUROPEAN JOURNAL, Issue 5 2008Abstract Enantiomerically pure, vicinal diols 1 afforded in a two-step synthesis (etherification and subsequent Claisen condensation) chiral bis-1,3-diketones H2L(S,S) (3,a,c) with different substitution patterns. Reaction of these C2 -symmetric ligands with various transition-metal acetates in the presence of alkali ions generated distinct polynuclear aggregates 4,8 by diastereoselective self-assembly. Starting from copper(II) acetate monohydrate and depending on the ratio of transition-metal ion to alkali ion to ligand, chiral tetranuclear copper(II) cubanes (C,C,C,C)-[Cu4(L(S,S))2(OMe)4] (4,a,c) or dinuclear copper(II) helicates (P)-[Cu2(L(S,S))2] (5) could be synthesized with square-pyramidal and square-planar coordination geometry at the metal center. In analogy to the last case, with palladium(II) acetate double-stranded helical systems (P)-[Pd2(L(S,S))2] (6,7) were accessible exhibiting a linear self-organization of ligand-isolated palladium filaments in the solid state with short inter- and intramolecular metal distances. Finally, the introduction of hexacoordinate nickel(II) in combination with lithium hydroxide monohydrate and chiral ligand H2L(S,S) (3,a) allowed the isolation of enantiomerically pure dinuclear nickel(II) coronate [(Li,MeOH)2,{(,,,)-Ni2(L(S,S))2(OMe)2}] (8) with two lithium ions in the voids, defined by the oxygen donors in the ligand backbone. The high diastereoselectivity, induced by the chiral ligands, during the self-assembly process in the systems 4,8 could be exemplarily proven by circular dichroism spectroscopy for the synthesized enantiomers of the chiral copper(II) cubane 4,a and palladium(II) helicate 6. [source] | |||||||||||||||||||||||||||||||