Metabolomics for the Quality Assessment of Lycium chinense Fruits

Aibellin is a 20-residue peptide antibiotic that has been isolated from the fungus Verticimonosporium ellipticum. Sequence-specific assignment of the ¹H- and ¹³C-NMR signals of aibellin in a methanol solution was achieved by using the two-dimensional NMR technique. Furthermore, its secondary structure was characterized by circular dichroism (CD) and NOESY spectra. The observed NOEs, ³J_NHCαH coupling constants and amide hydrogen–deuterium (H–D) exchange rates show that the peptide consisted of two α-helices and a bent structure around a Pro-14 residue.     

310-Helices,Helix Screw Sense and Screw Sense Reversal in the Dehydro-peptide Boc-Val-ΔPhe-Gly-ΔPhe-Val-OMe

The pentapeptide Boc-Val-ΔPhe-Gly-ΔPhe-Val-OMe, containing two dehydro-phenylalanine (ΔPhe) residues, has been synthesized and its structure investigated. In the crystalline state, the molecule adopts a right-handed 3₁₀-helical conformation stabilized by two intramolecular hydrogen bonds between CO of Val¹ and NH of ΔPhe⁴, and between CO of ΔPhe² and NH of Val⁵, respectively. NMR measurements are consistent with the presence of 3₁₀-helical structures also in acetonitrile and dimethylsulphoxide solution: the distances between backbone protons estimated from NOE connectivities are in overall agreement with those observed in the solid state; the chemical shifts of the amide protons show the smaller temperature coefficients for the NHs that in solid state are involved in intramolecular hydrogen bonds. The CD spectra in acetonitrile, chloroform, methanol and dimethylsulphoxide display exciton couplets of bands corresponding to the ΔPhe electronic transition at 280nm; the sign of the bands is consistent with the presence of helical structures having a prevalent left-handed screw sense. Addition of 1,1,1,3,3,3-hexafluoro- propan-2-ol gives rise to the gradual appearance of a couplet of opposite sign, suggesting the helix reversal from left-handed sense to right-handed sense. The conformational behaviour is discussed on the basis of the specific sequence of the peptide.     

Characterization of β-bend ribbon spiral forming peptides using electronic and vibrational CD

Terminally blocked (L-Pro-Aib)_n and Aib-(L-Pro-Aib)_n sequential oligopeptides are known to form right-handed β-bend ribbon spirals under a variety of experimental conditions. Here we describe the results of a complete CD and ir characterization of this subtype of 3₁₀-helical structure. The electronic CD spectra were obtained in solvents of different polarity in the 260-180 nm region. The vibrational CD and Fourier transform ir (FTIR) spectra were measured in deuterochloroform solution in the amide I and amide II (1750-1500 cm^?1) regions. The critical chain length for full development of the β-bend ribbon spiral structure is found to be five to six residues. Spectral effects related to concentration-induced stabilization of the structures of the longer peptides were seen in the resolution-enhanced FTIR spectra. Comparison to previous studies of (Aib)_n and (Pro)_n oligomers indicate that the low frequency of the amide I mode is due to the interaction of secondary and tertiary amide bonds and not to a strong difference in conformation from a regular 3₁₀-helix. © 1995 John Wiley & Sons, Inc.     

Influence of the peptide chain length on the stability of double-stranded β-helical species of D,L-alternating oligovalines in chloroform solution

The type and distribution of the β-helixes occurring in chloroform solutions of Boc-(L-Val-D-Val)₆-OMe and Boc-(L-Val-D-Val)₈-OMe have been studied by using ¹H-nmr techniques. Right- and left-handed ↑↓β^4.4-helices and left-handed β^5.6-helices occur with the dodecapeptide. β^4.4-Helices of opposite handedness occur also with the hexadecapeptide, but ↑↓β^5.6-helices could not be detected with this oligomer. At equilibrium, at 25°C, the double helix of the dodecapeptide is only moderately populated. These results indicate that increasing the chain length has a destabilizing effect on the ↑↓β^5.6-helices of D ,L -alternating oligovalines in chloroform solution.     

Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy

The energies of two and three-chain antiparallel and parallel β-sheets have been minimized. The chains were considered to be equivalent. In each case, chains consisting of four and of eight l-alanine residues, respectively, with CH₃CO- and -NHCH₃ end groups were examined. Computations were carried out both for chains constrained to have a regular structure (i.e. the same φ and ψ dihedral angles for each residue) and for chains in which the regularity constraint was relaxed. All computed minimum-energy β-sheets were found to have a right-handed twist, as observed in proteins. As in the case of right-handed α-helices, it is the intrastrand non-bonded interaction energy that plays the key role in forcing β-sheets of l-amino acid residues to adopt a right-handed twist. The non-bonded energy contribution favoring the right-handed twist is the result of many small pairwise interatomic interactions involving the C^βH₃ groups. Polyglycine β-sheets, lacking the C^βH₃ side-chains, are not twisted. The twist of the poly-l-alanine sheet diminishes as the number of residues per chain increases, in agreement with observations. The twist of the four-residue chain increases somewhat (because of interstrand non-bonded interactions, also involving the C^βH₃ groups) in going from a single chain to a two-chain antiparallel structure, but then decreases slightly in going from a two-chain to a three-chain structure. β-Sheets in observed protein structures sometimes have a larger twist than those in the structures computed here. This may be due to irregularities in amino acid sequence and in hydrogenbonding patterns in the observed sheets, or to long-range interactions in proteins. The minimized energies of parallel β-sheets are considerably higher than those of the corresponding antiparallel β-sheets, indicating that parallel β-sheets are intrinsically less stable. This finding about the two kinds of β-sheets agrees with suggestions based on analyses of β-sheets observed in proteins. The energy difference between antiparallel and parallel β-sheets is due to closer packing of the chains and a more favorable alignment of the peptide dipoles in the antiparallel structures. The hydrogen-bond geometry in the computed antiparallel structures is very close to that proposed by Arnott et al. (1967) for the β-form of poly-l-alanine.     

Solid state and solution structure of Boc-L-Ala-ΔPhe-ΔPhe-NHMe: A dehydropeptide showing propensity for 310-helices of both screw senses

The crystal and molecular structure of the peptide Boc-L -Ala-Δphe-Δphe-NHMe, containing two consecutive dehydro-phenylalanine (Δphe) residues, has been solved by x-ray diffraction. Two independent molecules, X and Y, are present in the crystallographic unit. Their conformation corresponds approximately to an incipient 3₁₀-helix stabilized by two intramolecular hydrogen bonds. The (?, ψ) torsion angles, however, have negative and positive signs in the two molecules X and Y, respectively. Therefore, in spite of the presence of an amino acid residue of the L configuration, the two helical molecules have opposite screw senses, even though the right-handed helix is less distorted than the left-handed one in correspondence of the L -Ala residue. The CD spectra in various solvents exhibit exciton bands originating from dipole–dipole interaction between the Δphe side chains. Addition of DMSO to the chloroform solution produces, as a first step, a strong increasing of the CD bands, which are then progressively canceled by increasing DMSO concentration. The nmr data parallel the behavior observed in the CD spectra. In CDCl₃ solution, the temperature coefficients of the NH resonances are consistent with the involvement of the last two amide protons of the sequence in intramolecular hydrogen bonds, but only negligibly small nuclear Overhauser effects (NOE) are observed. Addition of 5% DMSO-d₆ allows the observation of diagnostic NOEs. CD and nmr data indicate that the solid state structure is retained in solution, and are consistent with the presence of right-handed and left-handed conformers, with a prevalence of the more stable right-handed one. © 1993 John Wiley & Sons, Inc.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed C NMR

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the ¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting ¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴-10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However, ¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl ¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, ¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

Can PISEMA experiments be used to extract structural parameters for mobile β-barrels?

The effect of mobility on ¹⁵N chemical shift/¹⁵N–¹H dipolar coupling (PISEMA) solid state NMR experiments applied to macroscopically oriented β-barrels is assessed using molecular dynamics simulation data of the NalP autotransporter domain embedded in a DMPC bilayer. In agreement with previous findings for α-helices, the fast librational motion of the peptide planes is found to have a considerable effect on the calculated PISEMA spectra. In addition, the dependence of the chemical shift anisotropy (CSA) and dipolar coupling parameters on the calculated spectra is evaluated specifically for the β-barrel case. It is found that the precise choice of the value of the CSA parameters σ₁₁,σ₂₂ and σ₃₃ has only a minor effect, whereas the choice of the CSA parameter θ shifts the position of the peaks by up to 20 ppm and changes the overall shape of the spectrum significantly. As was found for α-helices, the choice of the NH bond distance has a large effect on the dipolar coupling constant used for the calculations. Overall, it is found that the alternating β-strands in the barrel occupy distinct regions of the PISEMA spectra, forming patterns which may prove useful in peak assignment.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-5094-5     

NMR solution structure of gramicidin A complex with caesium cations

The conformation of the 1:1 complex of [Val¹] gramicidin A with caesium cations has been determined in methanol/chloroform (1:1) solution by 2-dimensional ¹H-NMR spectroscopy. The molecular structure was found to be a right-handed antiparallel double helical dimer ↑↓ππ_LD^7.2 with 7.2 residues per turn, which incorporates two caesium cations.     

Secondary structure of peptides. 18. 15N-nmr and 13C-nmr CP/MAS investigation of the primary and secondary structure of (Ala/Val) copolypeptides

Various copolypeptides were prepared by benzylamine or tertiary amine-initiated copolymerizations of alanine–N-carboxyanhydride (Ala-NCA) and valine–N-carboxyanhydride (Val-NCA). The number-average molecular weights of these copolypeptides were detemined by ¹H-nmr spectroscopic end-group analyses and viscosity measurements. The sequences were characterized by ¹⁵N-nmr spectra in solution, and the average lengths of the homogeneous blocks were determined from the signal intensities. The 50.3-and 75.4-MHz ¹³C-nmr CP/MAS spectra of the solid copolypeptides are not sensitive to sequence effects, but allow qualitative and quantitative analyses of the secondary structures. In contrast to other methods, the ¹³C-nmr spectra allow determination of the extent to which individual amino acids are incorporated into β-sheet or α-helix phases. Depending on primary structure and molecular weight, the secondary structure of (Ala/Val) copolypeptides may vary significantly. Both monomer units may be predominantly helical or predominantly β-sheet structure, or the Val units may prefer the β-sheet structure with most Ala-units forming β-helices. However, these secondary structures are more or less thermodynamically unstable and revert to the stable conformations on reprecipitation from trifluoroacetic acid/water.     

Vibrational circular dichroism in amino acids and peptides. 7. Amide stretching vibrations in polypeptides

Vibrational circular dichroism (VCD) spectra for the principal amide stretching vibrations, amide A (N? H stretch) and amide I (predominantly C?O stretch), are presented and analyzed for a variety of polypeptides dissolved in chloroform, as well as for two examples in D₂O. Our results for poly(γ-benzyl-L -glutamate) confirm the first and only previous report of VCD in polypeptides carried out by Singh and Keiderling [(1981) Biopolymers 20 , 237–240]. Collectively, our spectra show that the sense of the bisignate VCD in these two regions depends on the sense of α-helicity and not on the absolute configuration of the constituent amino acids. This conclusion is established by obtaining VCD for the two polypeptides, poly(β-benzyl-L -asparate) and poly(im-benzyl-L -histidine), that form left-handed as opposed to right-handed α-helices. A new amide band having significant VCD intensity owing to its Fermi resonance interaction with the N? H stretching mode has been identified as a weak shoulder on the low-frequency side of the amide A band near 3200 cm^?1 and is assigned as a combination band of the amide I and amide II vibrations. VCD spectra of polypeptides in D₂O solution, although weak, have been successfully measured in the amide I region, where spectra appear to be more complicated due to the presence of solvated and internally hydrogen-bonded amide groups. Strong monosignate contributions to the VCD in the amide A and amide I regions for some of the polypeptides indicate coupling of an electronic nature between these two regions and is deduced by an application of the concept of local sum rules of rotational strength. It appears that a detailed understanding of the VCD obtained for polypeptides will not only be diagnostic of secondary structure, but also of more subtle structural and vibrational effects that give rise to local, intrinsic chirality in the amide vibrations.     

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, β-sheets, and 3₁₀-helices by strategically using ²H-labeled amino acids and SDSL. In this study, we have explored the use of ¹³C-labeled residues as the NMR active nuclei for this approach for the first time. ¹³C-labeled d₅-valine (Val) or ¹³C-labeled d₆-leucine (Leu) were substituted at a specific Val or Leu residue (i), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i-2 and i-3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The ¹³C ESEEM peaks in the FT frequency domain data were observed for the i-3 samples, and no ¹³C peaks were observed in the i-2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures.     

Secondary structures of peptides: 5 · 15N n.m.r. CP/MAS spectroscopy of solid polypeptides and gramicidin-S

30.5 MHz ¹⁵N m.m.r. (CP/MAS) spectra of various solid polypeptides were measured using the cross-polarization/magic angle spinning technique. In order to obtain optimum signal-to-noise ratios, relatively short contact times (1 ± 0.5 ms) are required, because the cross-polarization times (T_NH) are short and because the proton rotating-frame relaxation times (T_1p) are in the order of 20 ms. The ¹⁵N n.m.r. signals of copolypeptides may be sensitive to sequence effects; yet they are in most cases more sensitive to the nature of the secondary structure. The signals of α-helices absorb ca. 8–10 ppm upfield of β-sheet structures, whereas the polyglycine II helix absorbs downfield. The natural abundance spectrum of crystalline gramicidin-S exhibits a signal at ?247 ppm, a characteristic chemical shift of the antiparallel pleated sheet structure.     

1H-nmr studies of polyoxyethylene-bound homo-oligo-L-methionines

The use of ¹H-nmr spectroscopy is demonstrated to be a useful analytical method to characterize the structure of synthetic peptides attached to soluble, macromolecular polyoxyethylene (POE) supports in the liquid-phase method (LPM) of peptide synthesis. We report an extensive 360-MHz ¹H-nmr study of POE-bound homo-oligo-L -methionine peptides. A combination of high field and selective saturation or Redfield pulse methods allows resolution of individual backbone NH and α-CH resonances of dilute peptides in the presence of strong resonances from macromolecular POE and/or protonated solvents. The nmr spectra for the POE-bound peptides in CDCl₃ are qualitatively similar to those of the low-molecular-weight Boc-L -Met_n-OMe peptide esters. This corroborates other observations that POE has little effect on peptide stucture. The backbone α-CH region of peptides is overlapped by signals from the terminal oxyethylene group of POE, but the peptide side-chain and low-field backbone NH resonances are well resolved. In trifluoroethanol the Boc-(L -Met)_n-NH-POE heptamer and octamer adopt the right-handed α-helical structure, and the present nmr studies provide evidence for two strong intramolecular hydrogen bonds to stabilize the helices. In water, the N-deblocked derivatives, (L -Met)_n-NH-POE oligomers adopt β-sheet structure and manifest well-resolved nonequivalent NH resonances with 6–7 Hz ³J_NH-CH coupling constants.     

Preferential β-sheet structuration of multiconformational poly(Glu-Leu) by metallic ions

Summary Alternating poly(Glu-Leu) exhibits a random coil structure in pure water at neutral pH. The addition of 0.5 equiv of Ca²⁺ induces a coil-to-β-sheet transition and the addition of 0.15 equiv of Fe³⁺ induces a coil-to-α-helix transition. Conformational competition between these two structures was studied by mixing preformed β-sheets and α-helices in different proportions. Circular dichroism spectra clearly show that β-sheets are favored at the expense of α-helices in β-sheet-rich mixtures.     

FTIR spectroscopy of secondary-structure reorientation of melibiose permease modulated by substrate binding

Analysis of infrared polarized absorbance spectra and linear dichroism spectra of reconstituted melibiose permease from Escherichia coli shows that the oriented structures correspond mainly to tilted transmembrane α-helices, forming an average angle of ∼26° with the membrane normal in substrate-free medium. Examination of the deconvoluted linear dichroism spectra in H₂O and D₂O makes apparent two populations of α-helices differing by their tilt angle (helix types I and II). Moreover, the average helical tilt angle significantly varies upon substrate binding: it is increased upon Na⁺ binding, whereas it decreases upon subsequent melibiose binding in the presence of Na⁺. In contrast, melibiose binding in the presence of H⁺ causes virtually no change in the average tilt angle. The data also suggest that the two helix populations change their tilting and H/D exchange level in different ways depending on the bound substrate(s). Notably, cation binding essentially influences type I helices, whereas melibiose binding modifies the tilting of both helix populations.     

Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii

GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by ¹H-¹⁵N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobY^G153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobY^G153D:GTP complex was also found to be unstable over time.     

An aromatic schiff base stabilised as a coordinated enol iminium zwitterion by complex formation with a series of divalent metal ions

The Schiff base N-(2-hydroxy-3-carboxy-1-naphthylidine)-4-methyl-2-sulphonic acid aniline (bonsaH₃) has been found to react with a range of divalent metal ions (Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ and UO₂²⁺ to give red-yellow insoluble complexes (bonsaH)_m(H₂O)_n. The solid state diffuse reflectance spectra of all the complexes have an intense visible band at ca. 470 nm. This fact, together with evidence from infrared spectra and room-temperature magnetic-moment measurements, suggests that in all cases the ligand is coordinated to the metal ion in the solid state in the enol-iminium zwitterionic form. The ¹H NMR spectra of the Mg²⁺ and Zn²⁺ complexes in DMSO-d₆ indicate that a different structure is adopted in this solvent. Comparisons with the spectra of bonsa-H₃ and (bonsa-H₂)K·H₂O suggest that the solution structure is that of an enol-imine.     

Structural and mode of action studies on bio-active molecules

The technique of nuclear Overhauser effect difference spectroscopy allows the determination, from¹H nuclear magnetic resonance spectra, of those protons in a structure which are near in space to a selected, irradiated proton. The experiment is extremely powerful in the determination of structure in solution, and is sufficiently precise often to give stereochemical detail. The method was used in determination of the structures of the antibiotics of the teicoplanin complex (members of the vancomycin group), and the principles are briefly illustrated. Additionally, nuclear magnetic resonance pulse sequences can be used to edit¹³C spectra (separate the spectrum into four spectra, containing C, CH, CH₂, and CH₃ carbons), and this technique also aided the structure elucidation of the teicoplanin complex. Finally, it is emphasised that nuclear Overhauser effect difference spectroscopy can be used to determine the molecular details of drug binding sites, and an example is given.