Solution CD Spectra Research Articles

Mechanism of lysozyme adsorption onto gold surface determined by quartz crystal microbalance and surface plasmon resonance

In this study, the physicochemical characterization of lysozyme adsorbed on gold was investigated. Through the use of MP-SPR it was possible to establish that the orientation of molecules changes from side-on to between or end-on with increasing surface coverage. The data confirms that the process of adsorption is driven primarily by electrostatic interactions but also by hydrophobic forces. MP-SPR data was compared with the Random Sequential Adsorption model for a molecule with an ellipsoidal shape. Contact angle measurements showed that higher surface coverage also translates in more hydrophilic properties of obtained lysozyme layer. Comparison of CD and PM-IRRAS spectra in solution and adsorbed state respectively showed changes in the secondary structures of lysozyme. These changes are dependent on pH, but fundamentally they go in the direction of the increase of β-turn/random content with a simultaneous decrease in β-sheet fraction, which suggests that aggregation is not occurring. The combination of MP-SPR and QCM-D measurements allowed the estimation of the number of water molecules associated with the lysozymes films. It has been observed that hydration decreases from 70% in pH = 4 to 30% in pH = 11. This data indicates that hydration is driven mainly by the degree of protonation of lysozyme molecules.

Chiral 3D Coordination Polymers Consisting of Achiral Terpyridyl Precursors: from Spontaneous Resolution to Enantioenriched Induction.

A pair of enantiomers of three-dimensional (3D) chiral coordination polymers (CCPs) were successfully constructed by using achiral components 4,2':6',4''-terpyridyl precursors and Cu2+ through spontaneous resolution (1 a). By utilising feeding controlled chiral-templated induction and chiral auxiliary behaviour of optically pure camphor sulfonate (CSA), the enantioenriched (1 b-P and 1 b-M) and CSA captured (1 c-P and 1 c-M) CCPs were successfully synthesised, respectively. The chiral information of the corresponding products was confirmed by X-ray single crystal diffraction and solid-state CD spectra. Meanwhile, the formation processes of 1 b-P and 1 b-M were monitored through solution CD spectra, UV/Vis spectra and ESI-TOF MS. Based on these results, a reasonable chiral-templated induction mechanism of forming 1 b-P and 1 b-M was proposed.

Tuning the Structures of AsMo12 and AsW12 into Chiral Crystals by Introducing CH3CN and H2O

AbstractAbstract. The asymmetrical crystal [Na(15C5)2][Na(15C5)]2(AsMo12O40)]·CH3CN (1) was synthesized. Interestingly, the huge α‐keggin ion AsMo12 is distorted by introduction of CH3CN molecules and adopts a chiral assembly with space group P4. In contrast, the analogous compound AsW12 co‐crystallizes with trace H2O molecules. The hydrogen atoms of H2O are positionally disordered with 50 % occupation to correlate with AsW12 in space group P4/m. Solid and solution CD spectra indicate that the crystals of 1 are in R configuration. This leads to the conclusion that molybdenum has greater affinity toward organic CH3CN, whereas tungsten has greater affinity to inorganic H2O. Additionally, AsMo12 shows greater deformability than AsW12. It is suggested that POMo shows a greater potential as chiral material than POW because of the flexibility of Mo. As a highlight, CH3CN can function as a general chiral inducer for the innovation of other chiral crystals, if fixed along one dimension.

Synthesis of enantiomers of mononuclear Ru(II) complexes with 10,13-diaryl substituted dppz ligands

Abstract An array of enantiomerically pure mononuclcear [Ru(bpy) 2 (dppz)] 2 + derivatives with 10,13-diaryl substituted dppz ligand has been synthesized and characterized (bpy = bipyridine, dppz = pyrido-[3,2-a:2′,3′-c]phenazine). These new complexes exhibit substantially similar absorption spectra, resembling the parent complex [Ru(bpy) 2 (dppz)] 2 + , and the enantiomerically pure analogues show the similar CD spectra in buffer solution despite the structural difference.

Physicochemical characterisation of four peptide sequences related to thrombospondin-1B

Thrombospondin-1 (TSP-1) is a protein involved in angiogenesis and tumor metastasis. In a previous study, a tridecapeptide sequence of TSP-1B [KRFKQDGGWSHWG] was synthesized and its biological activity was determined as well as the activity of three related sequences TSPB-(E), TSPB-(S), and TSPB-(Abu) 6. These peptides were tested for activity on the cell growth of three human carcinoma cells lines and only TSPB-(Abu) 6 increased proliferation of MCF7 and HT-29. The main aim of this study was to perform physicochemical measurements, in a comparative way, to determine if the differences in activity could be related to physicochemical properties. Peptides were characterised by HPLC capacity factors, UV, fluorescence, and CD spectra (either in buffer solution or in the presence of lipid vesicles), surface activity, and aggregation. Moreover, the interaction of these peptides with phospholipids was determined through their penetration in monolayers of DPPC, PG, or PS as well as their miscibility in mixed monolayers. Besides, using liposomes as model membranes, the affinity of these peptides for phosphatidylcholine was measured with vesicles labeled with fluorescent markers (TMA-DPH, laurdan, pyrene). Results show that these molecules are highly hydrophilic and their surface activity is low. Mixed monolayers indicate that there is almost no miscibility. Besides, its presence does not modify noticeably the microviscosity of bilayers. Moreover, UV and fluorescence spectra of peptides were not affected by the presence of lipids in the media but CD spectra recorded in TFE/water (1/1) resulted in small changes for TSPB, TSPB-(E), and TSPB-(S) peptides. On the contrary CD spectra of TSPB-(Abu) 6 derivatives were clearly much more sensitive to the polarity of the environment. According to these data the biological activity of peptide with a cyclic aspartimide moiety at position 6 could be related to a specific conformational change in the peptide chain promoted by a hydrophobic membrane-like environment.

Membrane Interactions and Conformational Preferences of Human and Avian Prion N-Terminal Tandem Repeats: The Role of Copper(II) Ions, pH, and Membrane Mimicking Environments

The flexible N-terminal domain of the prion protein (PrP(c)) is believed to play a pivotal role in both trafficking of the protein through the cell membrane and its pathogenic conversion into the β sheet-rich scrapie isoform (PrP(sc)). Unlike mammalian PrP(c), avian prion proteins are not known to undergo any pathogenic conformational conversions. Consequently, some critical advances in our understanding of the molecular mechanisms underlying prion pathogenesis are expected from comparative studies of the biophysical properties of the N-terminal domains of the two proteins. The present study addresses the role played by different environmental factors, i.e., copper(II), pH, and membrane-mimicking environments, in assisting the conformational preferences of huPrP60-91 and chPrP53-76, two soluble peptides encompassing the N-terminal copper(II) binding domains of the human and chicken prion proteins, respectively. Moreover, the membrane interactions of huPrP60-91, chPrP53-76, and their copper(II) complexes were evaluated by Trp fluorescence in conjunction with measurements of the variation in thermotropic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. Circular dichroism experiments revealed that huPrP60-91 adopts a predominant polyproline II conformation in aqueous solution that is destabilized at basic pH or in the presence of trifluoroethanol (TFE). Unlike anionic sodium dodecyl sulfate (SDS), which seems to stabilize the polyproline II conformation further, zwitterionic dodecylphosphocholine (DPC) micelles do not affect the peptide structure. On the contrary, copper(II) promptly promotes an increase in β-turn-rich structures. Differential scanning calorimetry (DSC) and Trp fluorescence assays carried out on DPPC model membranes after incubation with huPrP60-91 showed a marked tendency of the peptide to slowly penetrate the lipid bilayer with a concomitant conformational transition toward an extended β-sheet-like structure. Such an event, which was ascribed to the hydrophobic Trp side chain residues, was shown to also depend on the level of copper(II) occupancy along the peptide. Conversely, the CD spectra of chPrP53-76 aqueous solutions indicated the presence of a mixture of random-coil/β-turn-like structures whose resulting equilibrium was influenced by SDS and copper(II) addition. Furthermore, chPrP53-76 did not exhibit any tendency to interact with model membranes in either the presence or absence of copper(II). The results reported here provide evidence of the different roles played by environmental factors in affecting the conformation and membrane activity of human and avian prion N-terminal domains.

Systematic Investigation of CD Spectra of Aryl Benzyl Sulfoxides Interpreted by Means of TDDFT Calculations

The CD spectra of 13 crystalline aryl benzyl sulfoxides 1a-m with various substituents on the two aromatic rings were recorded in solution and in the solid state. Solution CD spectra were very homogeneous along the series, consisting in most cases of a couplet-like feature in the 200-300 nm region. The red-shifted component of the couplet, corresponding to the sulfoxide-centered n-pi* transition, is always positive for (R) absolute configuration in accordance with Mislow's rule. The presence of a strong electron-withdrawing substituent on the phenyl ring (nitro or ester group) alters the shape of the CD spectrum. CD calculations using the TDDFT method were run for eight representative compounds using DFT-optimized geometries. In all cases, calculated spectra were in very good agreement with experimental ones and allowed for rationalization of the diverse spectral behaviors. It is demonstrated that TDDFT//DFT calculations represent a reliable option for assigning the absolute configuration of this class of compounds whenever crystals suitable for X-ray are not available. Solid-state CD spectra recorded with the KCl pellet technique were in some cases in agreement with those in solution. However, in other cases new and strong CD signals appeared which were interpreted as being due to intermolecular couplings in the crystals.

New Bioactive 2,3‐Epoxycyclohexenes and Isocoumarins from the Endophytic Fungus Phomopsis sp. from Laurus Azorica

AbstractSix new metabolites, namely cycloepoxylactone (1a), cycloepoxytriol A (2), cycloepoxytriol B (3) and phomolactones A–C (4–6), were isolated from two cultures of the ethyl acetate soluble fraction of the endophytic fungus Phomopsis sp. (internal strain no. 7233). Their structures were determined by means of spectroscopic data including HREIMS, 1H NMR, 13C NMR, and 2D NMR (HMQC, HMBC, and NOESY). The absolute configurations of 1a, 4 and 5 were determined by circular dichroism and comparison of solution CD spectra with TDDFT‐calculated ones. Preliminary studies showed that cycloepoxylactone (1a) has good antibacterial, antifungal, and algicidal activity against Bacillus megaterium, Microbotryum violaceum, and Chlorella fusca, respectively, whereas cycloepoxytriol B (3) has good algicidal activity against Chlorella fusca.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Circular dichroism of diglycosyl dichalcogenides in solution and solid state

Solution and solid-state CD spectra of nine peracetylated and deacetalyted diglycosyl disulfides were measured to study the relationship between the low-energy CD transitions (n1-->sigma*(S--S) and n2-->sigma*(S--S)) and helicity of the inherently chiral disulfide chromophore as perturbed by chiral carbohydrate moieties. The solid-state CD spectra were directly correlated with the reported X-ray structures of Ac4GlcSSGlcAc4 and Ac4GlcSSGalAc4, and the CD data revealed that all the disulfides have M helicity with C1--S--S--C1' dihedral angles -90 degrees < phi < 0 degrees both in solution and in the solid state. A TDDFT CD study was carried out on dimethyl diselenide which confirmed that the same quadrant rule is relevant between the signs of the low-energy CD transitions and the diselenide torsional angle as formulated previously for the disulfide chromophore. The CD spectra of Ac4GlcSeSeGlcAc4 measured in solution and in the solid state were correlated with its X-ray structure and reproduced well by TDDFT CD calculations performed on its tetra-O-acetyl derivative.

Crystal structure and chiroptical spectra of sodium tetrakis (+)-hfbc Pr(III) complex

Abstract The X-ray crystal structure of tetrakis (+)-hfbc Pr(III) complex, Na[Pr((+)-hfbc)4]·CH3CN, was compared with that of the corresponding Na–La complex with a pseudo-achiral dodecahedron, and the solution CD spectra were also discussed in the 4f–4f transition in consideration of the exciton CD couplet showing Δ-SAPR-8 configuration in CHCl3.

The Supramolecular Helical Architecture of 8‐Oxoinosine and 8‐Oxoguanosine Derivatives

The 8-oxoguanosine derivative 1 and the 8-oxoinosine derivative 2 b, with appropriate substituents on their ribose moieties, form hexagonal lyotropic mesophases in hydrocarbon solvents. Small-angle X-ray scattering analysis of a film of 1 and of the mesophase of 2 b, and NMR and CD spectra of isotropic solutions of 2 b, indicate that in both cases the supramolecular structures adopted are continuous helices formed by a hydrogen-bond network between the heterocyclic bases. Notably, while derivative 2 b, which bears large substituents on its ribose moiety, undergoes self-assembly and mesophase formation, oxoinosine 2 a, with only decanoyl groups on its ribose moiety, does not. This may be ascribed to the reduced amphiphilic properties of the latter and the absence of aromatic groups.

Novel Oxygen Chirality Induced by Asymmetric Coordination of an Ether Oxygen Atom to a Metal Center in a Series of Sugar-Pendant Dipicolylamine Copper(II) Complexes

Six sugar-pendant 2,2'-dipicolylamine (DPA) ligands (L1-3 and L'1-3) have been prepared. OH-protected and unprotected D-glucose, D-mannose, and D-xylose were attached to a DPA moiety via an O-glycoside linkage. X-ray crystallography of the copper(II) complexes (1-5) with these ligands revealed that the anomeric oxygen atom is coordinated to the metal center in the solid state, generating a chiral center at the oxygen atom. The CD spectra of these copper complexes in methanol or aqueous solution exhibit Cotton effects in the d-d transition region, which indicates that the ether oxygen atoms remain coordinated to the metal center and the oxygen-atom chirality is preserved even in solution. For complexes 1 and 2, the inverted oxygen-atom chirality and chelate-ring conformation in the solid state are well correlated with the mirror-image CD spectra in methanol solution. The concomitant inversion of the asymmetric configuration around the copper center was also observed in a methanol solution of complex 3 and a pyridine solution of complex 2. The square-pyramidal/octahedral copper(II) centers also exhibited characteristic absorption and CD spectra.

Conformational studies of oligomeric oxetane-based dipeptide isosteres derived fromL-rhamnose orD-xylose

Conformational investigations have been undertaken on oligomers (dimers, tetramers, hexamers) of five closely related oxetane-based dipeptide isosteres. All the oligomers were subjected to a range of studies by NMR, FT-IR and CD spectroscopy. The oligomers derived from methyl 2,4-anhydro-5-azido-3-O-tert-butyldimethylsilyl-5-deoxy-L-rhamnonate 'monomer' all exhibited evidence of ordered conformations in chloroform and 2,2,2-trifluoroethanol (TFE) solution. 5-Acetamido and N-methylamide derivatives of the L-rhamnonate 'monomer', along with a 'dimer' lacking silyl protection at C-3, were synthesized to ascertain the role of intramolecular interactions. This led to the conclusion that, for the L-rhamnonate oligomers, steric interactions govern the conformational preference observed. The equivalent silyl-protected D-lyxonate oligomers gave ordered CD spectra in TFE solution, but NMR and FT-IR spectroscopy in chloroform solution suggested an irregular, non-hydrogen bonded system. The remaining silyl-protected 6-deoxy-L-altronate, 6-deoxy-D-gulonate and D-fuconate oligomers appear to be characterized by their lack of ordered conformation in TFE and chloroform solution.

New Chromium Enolates

AbstractThree new (η2‐acrylate)(η6‐arene)dicarbonylchromium complexes are reported. They were obtained either by CO/acrylate exchange in [Cr(η6‐benzene)(CO)3] (1) via the photolytically generated η2‐cyclooctene intermediate or by arene exchange in [Cr(η2‐methyl acrylate)(η6‐benzene)(CO)2] (3) (Scheme 1). On crystallization, [Cr(η2‐methyl acrylate)(η6‐o‐xylene)(CO)2] (5) underwent partial resolution. The degree of this resolution was analyzed via X‐ray crystal‐structure determination (Fig. 1) and was correlated to the CD spectra in solution (Fig. 6), thus allowing the assignment of the absolute configuration. The reaction of [Cr(η2‐acrylate)(η6‐benzene)(CO)2] complexes with cyclopentad‐1,3‐diene or 1H‐indene afforded new (η6‐cyclopenta‐2,4‐dien‐1‐yl)‐ or (η6‐1H‐inden‐1‐yl)(η3‐oxaallyl)chromium complexes (Scheme 2). A mechanism is proposed that involves arene‐ligand substitution by the diene ligand, initiated by haptotropic rearrangement of the acrylate from a η2‐ to a η4‐coordination mode, followed by hydride migration from the diene to the acrylate (Scheme 3). An X‐ray crystal‐structure determination of [Cr(CO)2(η5‐1H‐inden‐1‐yl){η3‐CH(CH2CF3)C(O)OEt}] (8) reveals a metal enolate that is best described as η3‐oxaallyl species (Fig. 2). A shorter, more‐efficient route to the [Cr(η5‐1H‐inden‐1‐yl)(η3‐oxaallyl)] complexes was devised via the reaction of [Cr(CO)2(η2‐cyclooctene)(η6‐1H‐indene)] with methyl acrylate (Scheme 4).

Solid‐Phase Synthesis of a β3‐Icosapeptide Containing the Homologs of the Twenty Common Proteinaceous Amino Acids. Preliminary Communication

AbstractAn icosapeptide, 1, containing the β3‐amino acid residues with the 20 proteinogenic side chains has been assembled by manual solid‐phase synthesis, according to the Fmoc strategy. The sequence was chosen in such a way that a possible 314‐helical conformation (secondary structure) would be stabilized by salt bridges and have an amphipathic character (Fig. 1,a), and the N‐terminal β3hCys would lend itself to thioligations and disulfide formation (2 and 3, in Figs. 1 and 2). The products 1–3 were pure according to RP‐HPLC, NMR, and MS analysis (Fig. 1,b and c, Fig. 2,c and d, and Fig. 3). With due caution, the CD spectra in aqueous solution (pH 7) and in MeOH (Fig. 4), with normalized Cotton effects θ =−14000 to −16000 [deg⋅cm2⋅dmol−1] between 209 and 210 nm, might be taken as an evidence for the presence of 314‐helical conformations. An evaluation of the data from a 700‐MHz 2D‐NMR measurement of the disulfide 2 in CD3OH is in progress.

Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers

The proteins PLM (phospholemman), CHIF (channel inducing factor), and Mat8 (mammary tumor protein 8 kDa) are members of the FXYD family of ion transport regulatory membrane proteins. Here we describe their cloning and expression in Escherichia coli, and their purification for NMR structural studies in lipid micelles and lipid bilayers. The molecular masses of the purified recombinant FXYD proteins, determined from SDS-PAGE and from MALDI TOF mass spectrometry, reflect monomeric species. The solution NMR and CD spectra in SDS micelles show that they adopt helical conformations. The solid-state NMR spectra in lipid bilayers give the first view of their transmembrane architecture.

Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT(1A) receptor.

Homology modeling of the angiotensin II AT(1A) receptor based on rhodopsin's crystal structure has assigned the 92-100 (YRWPFGNHL) sequence of the receptor to its first extracellular loop. Solution and membrane-bound conformational properties of a peptide containing this sequence (EL1) were examined by CD, fluorescence, and (1)H-NMR. CD spectra in aqueous solution revealed an equilibrium between less organized and folded conformers. NMR spectra indicated the coexistence of trans and cis isomers of the Trp(3)-Pro(4) bond. A positive band at 226 nm in the CD spectra suggested aromatic ring stacking, modulated by EL1's ionization degree. CD spectra showed that trifluoroethanol (TFE), or binding to detergent micelles and phospholipid bilayers, shifted the equilibrium toward conformers with higher secondary structure content. Different media gave rise to spectra suggestive of different beta-turns. Chemical shift changes in the NMR spectra corroborated the stabilization of different conformations. Thus, environments of lower polarity or binding to interfaces probably favored the formation of hydrogen bonds, stabilizing beta-turns, predicted for this sequence in the whole receptor. Increases in Trp(3) fluorescence intensity and anisotropy, blue shifts of the maximum emission wavelength, and pK changes also evinced the interaction between EL1 and model membranes. Binding was seen to depend on both hydrophobic and electrostatic interactions, as well as lipid phase packing. Studies with water-soluble and membrane-bound fluorescence quenchers demonstrated that Trp(3) is located close to the water-membrane interface. The results are discussed with regard to possible implications in receptor folding and function.

Isolation, purification, and characterization of catalase from the methylotrophic yeast Pichia pastoris.

Catalase (CATpp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration. Quantitative parameters of absorption and CD spectra of CATpp solutions and of its membrane-concentrated form (CATpp-conc) were studied. Rates of H2O2 decomposition and kinetic characteristics Km and kcat of CATpp and CATpp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30 degrees C, as well as the effective constant kin of the enzyme inactivation rate during the catalysis and the constant k2 of the interaction rate of the Complex I catalases with H2O2. Thermal inactivation of CATpp in solutions at 45 degrees C was characterized by the effective rate constant kin*, and the low-frequency (27 kHz) ultrasonic inactivation of CATpp at 20 degrees C was characterized by the first-order rate constant kin(US). All spectral and kinetic characteristics of CATpp and CATpp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CATcb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CATpp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of kcat/Km, thermal stability comparable with the thermal stability of CAT in terms of kin*, the minimal kin, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm2.

Reversible peptide folding: Dependence on molecular force field used

Temperature-dependent nuclear magnetic resonance (NMR) and CD spectra of methanol solutions of a β-heptapeptide have been interpreted in such a way that the secondary structure, a 314-helix, is assumed to be stable in a temperature range of between 298 and 393 K. This is in contrast to the results of a 50-ns molecular dynamics simulation using the GROMOS96 force field, which found a melting temperature of about 340 K. This discrepancy is addressed by further computational studies using the OPLS-AA force field. The conformational energetics of N-formyl-3-aminobutanamide in vacuo are obtained using ab initio and density functional quantum-mechanical calculations at the HF/6-31G*, B3LYP/6-31G*, and B3LYP/6-311+G* levels of theory. The results permit development of torsional parameters for the OPLS-AA force field that reproduce the conformational energetics of the monomer. By varying the development procedure, three parameter sets are obtained that focus on reproducing either low-energy or high-energy conformations. These parameter sets are tested by simulating the reversible folding of the β-heptapeptide in methanol. The melting temperature of the helix formed (>360 K) is found to be higher than the one obtained from simulations using the GROMOS96 force field (∼340 K). Differences in the potential energy functions of the latter two force fields are evaluated and point to the origins of the difference in stability. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 774–787, 2000

The structure of the membrane-binding 38 C-terminal residues from bovine PP3 determined by liquid- and solid-state NMR spectroscopy.

The secondary structure and membrane-associated conformation of a synthetic peptide corresponding to the putative membrane-binding C-terminal 38 residues of the bovine milk component PP3 was determined using 1H NMR in methanol, CD in methanol and SDS micelles, and 15N solid-state NMR in planar phospholipid bilayers. The solution NMR and CD spectra reveal that the PP3 peptide in methanol and SDS predominantly adopts an alpha-helical conformation extending over its entire length with a potential bend around residue 19. 15N solid-state NMR of two PP3 peptides 15N-labelled at the Gly7 and Ala32 positions, respectively, and dissolved in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol phospholipid bilayers shows that the peptide is associated to the membrane surface with the amphipathic helix axis oriented parallel to the bilayer surface.