Estimation of pseudopeptide metal ion complexation tendencies by special MS methods,HPCE and circular dichroism

Summary To obtain more insight into catalytic mechanisms of metallo enzymes and specific metal complexation by proteins we use linear and cyclic pseudopeptides as mimetics. Knowledge about tendencies of complex formation of different ligands with selected transition metal ions is an indispensable prerequisite for the development of homo-and hetero-dinuclear metallo enzyme mimetics. Three pseudotripeptide ligands were investigated with respect to formation tendency and properties of complexes with the transition metal ions Cu²⁺, Zn²⁺ Ni²⁺, Co²⁺ and Mn²⁺. To study complexation tendencies we applied different methods. One of the important prerequisites for the application in a secreening of series of peptide ligands is the necessity for a minimal amount of substance. We used and compared certain masspectrometric methods for the estimation of a rank order of complexation of certain transition metal ions. We also applied spectrophotometric titration, circular dichroism measurements, capillary electrophoresis and pH-rate profile of catalytic activity in the attempt to evaluate complex formation tendencies. Except for the spectrophotometric pH-titration and the pH-profile of catalytic activity all methods, were applicable, but each method has its advantages and disadvantages depending on the separation effect of the ligand from the metal complex, and depending on the spectroscopic properties of ligand and complex. The results regarding complex formation are compared to each other. Comparison of pairs by MALDI-TOF-and ESI-MS allows an estimation of the rank order of complexation tendency of one ligand with different metal ions and requires the least amount of substance. The other investigated methods provided additional information on structural properties of the formed complexes; however either they required too much pseudopeptide ligand or were not applicable for all transition metal ions used in this study.     

Derivatization,complexation, and absolute configurational assignment of chiral primary amines: application of exciton-coupled circular dichroism

We report here a sensitive method for the determination of the absolute configurations of primary amines using exciton-coupled circular dichroism (ECCD). The method works on a microgram scale by derivatization of chiral amines with quinoline chromophores. Complexation of the chiral ligands with metal ion fixes the geometry of the chromophores, resulting in a twist that is governed by the asymmetric carbon configuration and steric environment of the amine. The absolute configurations of the primary amines can be interpreted from the couplets of the ECCD spectra of the derivatized complexes. Crystal structures, 2D NMR studies, and semiempirical calculations provide structural evidence for our model.     

Complexation of metal ions by pseudotripeptides with different functionalized N-alkyl residues

Summary Seven pseudotripeptides with the common structure Bz-His-ψ[CO−N(CH₂)n-X]Gly-His-NH₂ were synthesized on the solid phase using the Fmoc-strategy, trityl protection for both His residues and Boc-or-OBu^t-protection for N-aminoalkyl-and N-carboxyalkyl residues, respectively. Functionalized N-alkyl glycyl peptides were formed on the solid phase by amination of a bromoacetyl dipeptide. All seven pseudotripeptides are able to form chelate complexes with the metal ions Zn²⁺, Ni²⁺, Cu²⁺ and Co²⁺. The existence of monomeric 1∶1 complexes for these pseudopeptides was calculated from the MW estimated by MALDI-MS and from the isotope distribution pattern estimated by ESI.     

Complexation of metal ions by pseudotripeptides with different functionalized N-alkyl residues

Seven pseudotripeptides with the common structure Bz-His-[CO-N(CH₂)n-X]Gly-His-NH₂ were synthesized on the solid phase using the Fmoc-strategy, trityl protection for both His residues and Boc- or -OBu^t-protection for N-aminoalkyl- and N-carboxyalkyl residues, respectively. Functionalized N-alkyl glycyl peptides were formed on the solid phase by amination of a bromoacetyl dipeptide. All seven pseudotripeptides are able to form chelate complexes with the metal ions Zn²⁺, Ni²⁺, Cu²⁺ and Co²⁺. The existence of monomeric 1:1 complexes for these pseudopeptides was calculated from the MW estimated by MALDI-MS and from the isotope distribution pattern estimated by ESI.     

Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

Secondary structural analysis of retrovirus integrase: characterization by circular dichroism and empirical prediction methods

The retrovirus integrase (IN) protein is essential for integration of viral DNA into host DNA. The secondary structure of the purified IN protein from avian myeloblastosis virus was investigated by both circular dichroism (CD) spectroscopy and five empirical prediction methods. The secondary structures determined from the resolving of CD spectra through a least-squares curve fitting procedure were compared with those predicted from four statistical methods, e.g., the Chou-Fasman, Garnier-Osguthorpe-Robson, Nishikawa-Ooi, and a JOINT scheme which combined all three of these methods, plus a pure a priori one, the Ptitsyn-Finkelstein method. Among all of the methods used, the Nishikawa-Ooi prediction gave the closest match in the composition of secondary structure to the CD result, although the other methods each correctly predicted one or more secondary structural group. Most of the alpha-helix and beta-sheet states predicted by the Ptitsyn-Finkelstein method were in accord with the Nishikawa-Ooi method. Secondary structural predictions by the Nishikawa-Ooi method were extended further to include IN proteins from four phylogenetic distinct retroviruses. The structural relationships between the four most conserved amino acid blocks of these IN proteins were compared using sequence homology and secondary structure predictions.     

Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone.     

Composition and metal ion complexation behavour of humic fractions derived from corn tissue

Decomposition of fresh plant residues produces humic fractions with different molecular size and composition. It was hypothesized that the functional group-type and content of humic fractions depended on molecular size, which was expected to influence heavy-metal complexation behavior. In this study, corn (Zea maysL.) stalks and leaves were collected from the field and decomposed for an 8-month period to produce humic substances which were separated into three water soluble fractions, HF1, HF2 and HF3, from highest to lowest relative molecular size. Functional group determination showed that total, carboxylic and phenolic OH acidity increased as relative molecular size of humic fractions decreased. Furthermore, C/O ratios decreased, whereas N/C and H/C ratios remained relatively unaffected as relative molecular size of humic fractions decreased. Formation of Ca²⁺, Cd²⁺ and Cu²⁺ -humic fraction complexes and how these complexes were affected by pH and relative (humic fraction) molecular size were studied using potentiometric titration. Metal-humic complexes exhibited at least two types of sites with respect to Ca²⁺, Cd²⁺ and Cu²⁺ complexation. Relative molecular size had a large significant influence on total metal-ion complexation, but it had a relatively small influence on complex stability at low levels of metal-ion complexation. Strength of metal-ion humic complexes followed the order Cu²⁺ > Cd²⁺ > Ca²⁺ and was affected by pH, especially for low affinity sites. Carboxylic and phenolic OH groups were most likely involved in complex formation. Magnitude of the metal-humic formation constants at the lowest equilibrium metal-ion concentration, under the various pH values tested, varied from 5.39 to 5.90 for Ca²⁺, from 5.36 to 6.01 for Cd²⁺ and from 6.93 to 7.71 for Cu²⁺. Furthermore, the formation constants appeared to be positively influenced by decreasing molecular size of water-soluble humic fraction, and increasing pH. These results inferred that soil management practices causing build-up of humic substances would affect mobility and bioavailability of metal-ions.     

Comparative analysis of inosine-substituted duplex DNA by circular dichroism and X-ray crystallography

Leveraging structural biology tools, we report the results of experiments seeking to determine if the different mechanical properties of DNA polymers with base analog substitutions can be attributed, at least in part, to induced changes from classical B-form DNA. The underlying hypothesis is that different inherent bending and twisting flexibilities may characterize non-canonical B-DNA, so that it is inappropriate to interpret mechanical changes caused by base analog substitution as resulting simply from ‘electrostatic’ or ‘base stacking’ influences without considering the larger context of altered helical geometry. Circular dichroism spectra of inosine-substituted oligonucleotides and longer base-substituted DNAs in solution indicated non-canonical helical conformations, with the degree of deviation from a standard B-form geometry depending on the number of I?C pairs. X-ray diffraction of a highly inosine-substituted DNA decamer crystal (eight I?C and two A?T pairs) revealed an A-tract-like conformation with a uniformly narrow minor groove, reduced helical rise, and the majority of sugars adopting a C1′-exo (southeastern) conformation. This contrasts with the standard B-DNA geometry with C2′-endo sugar puckers (south conformation). In contrast, the crystal structure of a decamer with only four I?C pairs has a geometry similar to that of the reference duplex with eight G?C and two A?T pairs. The unique crystal geometry of the inosine-rich duplex is noteworthy given its unusual CD signature in solution and the altered mechanical properties of some inosine-containing DNAs.     

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.     

Electronic structures of azulene-fused porphyrins as seen by magnetic circular dichroism and TD-DFT calculations

A combination of magnetic circular dichroism (MCD), electronic absorption spectroscopy and time-dependent density functional theory (TD-DFT) calculations has been used to investigate the electronic structure of azulene-fused pi-expanded porphyrins based primarily on the spectral properties of absorption bands in the near infrared region. From MCD experiments, it was suggested that in the case of a mono-azulene-fused porphyrin DeltaHOMO approximately equal DeltaLUMO (where DeltaHOMO is the magnitude of the energy gap between the HOMO and HOMO-1 and DeltaLUMO is the magnitude of the energy gap between the LUMO and LUMO+1), while in the case of an oppositely-di-azulene-fused porphyrin, DeltaHOMO相似文献   

Conformational studies of wheat flour high relative molecular mass glutenin subunits by circular dichroism spectroscopy

Conformational studies of 1Dx2, 1Bx7, and 1Dy12 high relative molecular mass glutenin subunits, extracted from Alisei 1 flour, are reported. Circular dichroism (CD) spectroscopy is employed to study their conformational polymorphism induced by urea and by urea in the presence of 1% sodium dodecyl sulfate (SDS). The CD spectra indicate that SDS promotes ordered structures. The addition of urea to the SDS-acetate solution of 1Dx2, 1Bx7, and 1Dy12 subunits eliminates the effect of SDS. Its addition to the acetate solution of proteins induces conformational transitions to form a poly-L-proline II-like structure. All the changes induced by urea follow a multistep transition process that is typical of proteins consisting of different domains.     

Formation and temperature stability of G-quadruplex structures studied by electronic and vibrational circular dichroism spectroscopy combined with ab initio calculations

Variations in the structure of d(GGGA)(5) oligonucleotide in the presence of Li(+), Na(+), and K(+) ions and its temperature stability were studied using electronic and vibrational circular dichroism, IR absorption, and ab initio calculations with the Becke 3-Lee-Yang-Parr functional at the 6-31G** level. The samples were characterized by nondenaturing gel electrophoresis. Oligonucleotide d(GGGA)(5) in the presence of Li(+) forms a nonplanar single tetramer, with angles of 102 degrees and 171 degrees between neighboring guanine bases. This tetramer changes its geometry at temperatures >50 degrees C, but does not form a quadruplex structure. In the presence of Na(+), the d(GGGA)(5) structure was optimized to almost planar tetramers with an angle of 177 degrees between neighboring guanines. The spectral results suggest that it stacks into a quadruplex helical structure. This quadruplex structure decayed to a single tetramer at temperatures >60 degrees C. The Hartree-Fock energies imply that d(GGGA)(5) prefers to form complexes with Na(+) rather than Li(+). The d(GGGA)(5) structure in the presence of monovalent ions is stabilized against thermal denaturation in the order Li(+) < Na(+) < K(+).     

Studies of human transcortin at different pHs: circular dichroism, polymerisation and binding affinity.

The transcortin we have used in this work is extremely pure. This was shown by the polymerisation observed at pH 4. This polymerisation is never observed with an impure form of transcortin [4]. Moreover, since it is known that the presence of cortisol in the binding site is an essential condition to the activity of purified transcortin [5], it appears that a correlation between the secondary structure and the biological activity of the transcortin exists. The results we have obtained are summarized below: (1) The inhibition of the transcortin binding capacity essentially takes place between pH 5 and 4. (2) A reorganisation of the structure of the protein moiety is observed between pH 6.5 and 5.9. (3) A decrease of the helicity ratio is observed between pH 5 and 4. It appears therefore that, in the limits of experimental accuracy of CD measurements to determine the amount of beta-structure, no appreciable change of binding activity is taking place after the appearance of a large percentage of beta-structure between pH 6.5 and 6. On the other hand, the sudden decrease of protein activity at low pH is likely to be correlated with the disappearance of a well-defined helical region. Other biochemical and physical experiments would be of course necessary, in order to precise this first observation of a structure-function relationship in transcortin.     

Determination of the absolute configuration and solution conformation of gossypol by vibrational circular dichroism

Vibrational circular dichroism (VCD) measurements and density functional theory (DFT) calculations were used to obtain the first definitive assignment of the absolute configuration for the polyphenolic binaphpthyl dialdehyde gossypol and a determination of the solution conformation in CDCl(3). VCD spectra recorded for the two resolved enantiomers are near mirror images and excellent agreement between the observed IR and VCD spectra and intensity calculations carried out at the DFT (B3LYP/6-31G*) level establish the absolute configurations of (+)-gossypol as P and (-)-gossypol as M, with two conformations in CDCl(3) solution that differ in isopropyl group orientation.     

Complexation of a four-strand tetraalcohol with labile metal ions probed by electrospray mass spectrometry

The saturated, stereodefined tetraalcohol 2,3,5,6-endo,endo,endo,endo-tetrakis(hydroxymethyl)bicyclo[2.2.1]heptane (tetol, L¹) and the simple alcohol butane-1,3-diol (L²) form complexes with alkali metal ions (lithium, sodium, potassium, rubidium and caesium), alkali earth cations (magnesium, calcium, strontium and barium) and Ga(III) and Ce(IV) in aqueous solution, characterised by electrospray ionisation mass spectrometry (ESMS). Metal ion exchange between the Li⁺ complex of L¹ and the other metal ions is rapid, with a range of M(L¹)_n ^m+ species detected, in addition to solvated species. With the alkal metal ions, M(L¹)⁺ and M(L¹)₂ ⁺ are dominant, although speciation varies with metal ion size. For the alkaline earth ions, a range of complex ions up to n=8 are observed, although n=1-3 dominate. A preference for M(L¹)₂ ²⁺ with Mg²⁺ versus M(L¹)₃ ²⁺ with Ca²⁺ may again relate to a larger ion size. For the higher-charged Ga(III) and Ce(IV), hydroxo species M(OH)(L¹)_n ^(m−1)+ are dominant reflecting bulk solution behaviour, which the ESMS studies appear to map generally.     

Chiral dialkyl ditellurides: Conformation of chiral chromophore by circular dichroism and DFT calculation

The twisted structure of ditellurides, in a similar way as in other dichalcogenes, leads to different absorption of circularly polarized light by quasi‐enantiomeric chiral orbitals. Chiral optically active ditellurides are not common compounds and this phenomenon is not widely reported. As chiral ditellurides found an application in asymmetric synthesis, their molecular structure, understood as their conformation, became an important factor for understanding their reactivity. Until now there are few examples of chiral ditellurides known and their structure was not analyzed in details. This article presents the results of our most recent research on the structure of chiral ditellurides investigated by electronic circular spectroscopy (ECD) supported by quantum‐chemical calculation. This enables us to suggest a relationship between chirality of alkyl substituent and chirality (conformation) of ditelluride.     

Reliable HPLC separation,vibrational circular dichroism spectra,and absolute configurations of isoborneol enantiomers

Resolution of chiral compounds has played an important role in the pharmaceutical field, involving detailed studies of pharmacokinetics, physiological, toxicological, and metabolic activities of enantiomers. Herein, a reliable method by high‐performance liquid chromatography (HPLC) coupled with an optical rotation detector was developed to separate isoborneol enantiomers. A cellulose tris(3, 5‐dimethylphenylcarbamate)‐coated chiral stationary phase showed the best separation performance for isoborneol enantiomers in the normal phase among four polysaccharide chiral packings. The effects of alcoholic modifiers and column temperature were studied in detail. Resolution of the isoborneol racemate displayed a downward trend along with an increase in the content of ethanol and column temperature, indicating that less ethanol in the mobile phase and lower temperature were favorable to this process. Moreover, two isoborneol enantiomers were obtained via a semipreparative chiral HPLC technique under optimum conditions, and further characterized by analytical HPLC, and experimental and calculated vibrational circular dichroism (VCD) spectroscopy, respectively. The solution VCD spectrum of the first‐eluted component was consistent with the Density Functional Theory (DFT) calculated pattern based on the SSS configuration, indicating that this enantiomer should be (1S , 2S , 4S )‐(+)‐isoborneol. Briefly, these results have provided reliable information to establish a method for analysis, preparative separation, and absolute configuration of chiral compounds without typical chromophoric groups.     

Measurement of circular birefringence and circular dichroism of the single crystals of lambda-(+)589- and delta-(-)589-tris(ethylenediamine)cobalt(III) triiodide monohydrate by the extended HAUP method

We have achieved measuring four optical parameters simultaneously, namely, linear birefringence (LB), circular birefringence (CB), linear dichroism (LD), and circular dichroism (CD), of single crystals of Lambda-(+)(589)- and Delta-(-)(589)-tris(ethylenediamine) cobalt(III) triiodide monohydrate (1) along the <001> plane at the fixed wavelength (514.5 nm). Such measurements are possible only when the High Accuracy Universal Polarimeter (HAUP) is employed; it is called the extended HAUP method. Our experimental results showed that both LB and LD of the Lambda-(+)(589)-(1) crystal have the same magnitude as those of the Delta-(-)(589)-(1) crystal. It was also revealed for the first time that the CB data of crystals of Lambda-(+)(589)-(1) and Delta-(-)(589)-(1) are almost of the same magnitude, but are of opposite sign, reflecting their opposite absolute configurations. On the other hand, although the CD data obtained for Lambda-(+)(589)-(1) is almost three times larger than that for Delta-(-)(589)-(1,) these CD data are also opposite in sign, as expected from the opposite chirality of crystals. .     

Determination of the absolute configuration and solution conformation of a novel disubstituted pyrrolidine acid A by vibrational circular dichroism

Compound A, a novel disubstituted pyrrolidine acid, is a member of a new class of agents that are potentially useful for the treatment of diabetes and dyslipidemia. The absolute configuration of this compound was determined by using vibrational circular dichroism (VCD). The results are in agreement with the assignments based on both X-ray analysis and the stereo-selective chemical synthesis. During VCD analysis, the solution conformation for a portion of compound A in CDCl(3) was also established. The compound is found to associate as an H-bonded carboxylic acid "dimer" in CDCl(3) solution, and VCD calculations on a model dimer fragment were required to establish the absolute configuration.