Spatial structure of angiotensin in an aqueous solution

The spatial structure of spin-labeled angiotensin in aqueous solution wa investigated with the combined use of NMR, fluorescence spectroscopy and energy calculation including Monte-Carlo techniques. The calculated mean values of molecular parameters were compared with the experimental ones. The calculated and experimental mean values were regarded as statistically indistinguishable when the corresponding mean values occurred within the 95% confidence limit. The experimental parameters were shown to be adequately described by calculated conformers only with the assumption of the existence of dynamic equilibrium of conformers in solution. The mean values of statistical weights and their limits providing the agreement between the calculated and experimental data were determined. Two geometrically different forms of backbone structure for C-terminal hexapeptide in aqueous solution were revealed using the discussed approach; the N-terminal part of the molecule appeared to be much more conformationally labile. The model of molecule spatial structure is consistent with available literature data upon angiotensin titration experiments, its complexing with heavy metal ions etc.     

The structure of gellan in dilute aqueous solution

A full assignment of high-field nmr spectra of gellan was obtained in dilute aqueous solution by performing a series of selective one-dimensional nmr experiments. The observed nuclear Overhauser effects (NOEs) cannot be interpreted assuming that each sugar residue is intrinsically rigid and in a chair conformation. In fact, the rhamnose residue gives strong NOE contacts coherent only with an equilibrium involving both a chair as well as a boat (or a hemiboat) conformation. Molecular dynamic calculations performed on a heptamer with a central rhamnose support the above finding, and show a structure based on a very stiff single chain in which it is present a flipping of the rhamnose residue. At low temperatures (5-20 degrees C) in very dilute solutions (0.018 mg/mL) nmr spectra show a splitting of the resonance due to the methyl group of rhamnose residue, thus confirming the presence of a slow equilibrium among different conformers.     

The three-dimensional solution structure of mini-M conotoxin BtIIIA reveals a disconnection between disulfide connectivity and peptide fold

Conotoxins are bioactive peptides from the venoms of marine snails and have been divided into several superfamilies based on homologies in their precursor sequences. The M-superfamily conotoxins can be further divided into five branches based on the number of residues in the third loop of the peptide sequence. Recently two M-1 branch conotoxins (tx3a and mr3e) with a C1–C5, C2–C4, C3–C6 disulfide connectivity and one M-2 branch conotoxin (mr3a) with a C1–C6, C2–C4, C3–C5 disulfide connectivity were described. Here we report the disulfide connectivity, chemical synthesis and the three-dimensional NMR structure of the novel 14-residue conotoxin BtIIIA, extracted from the venom of Conus betulinus. It has the same disulfide connectivity as mr3a, which puts it in the M-2 branch conotoxins but has a distinctly different structure from other M-2 branch conotoxins. 105 NOE distance restraints and seven dihedral angle restraints were used for the structure calculations. The three-dimensional structure was determined with CYANA based on torsion angle dynamics and refinement in a water solvent box was carried out with CNS. Fifty structures were calculated and the 20 lowest energy structures superimposed with a RMSD of 0.49 ± 0.16 Å. Even though it has the M-2 branch disulfide connectivity, BtIIIA was found to have a ‘flying bird’ backbone motif depiction that is found in the M-1 branch conotoxin mr3e. This study shows that conotoxins with the same cysteine framework can have different disulfide connectivities and different peptide folds.     

Two-dimensional NMR studies and secondary structure of cobrotoxin in aqueous solution

The 1H-NMR spectra of cobrotoxin, a neurotoxic protein isolated from Formosan cobra Naja naja atra, have been studied by two-dimensional NMR techniques. Of 62 amino acid residues in cobrotoxin, the complete assignments of 58 residues have been made. The resonances from several of the remaining residues have been identified but not yet specifically assigned. The secondary structure of an antiparallel triple- and double-stranded beta-sheet has also been determined by observing the NOE.     

Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl-tRNA synthetase

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl-tRNA synthetase. The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and T psi arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPhe since beef tRNATrp showed a more stable D stem and a less stable T psi stem. When alkylation by ethylnitrosourea was studied with the tRNATrp X tryptophanyl-tRNA synthetase complex we found that phosphates located at the 5' side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N-3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase X tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl-tRNA synthetase was confirmed in nuclease S1 mapping experiments.     

Quaternary structure of the neuronal protein NAP-22 in aqueous solution

NAP-22, a myristoylated, anionic protein, is a major protein component of the detergent-insoluble fraction of neurons. After extraction from the membrane, it is readily soluble in water. NAP-22 will partition only into membranes with specific lipid compositions. The lipid specificity is not expected for a monomeric myristoylated protein. We have studied the self-association of NAP-22 in solution. Sedimentation velocity experiments indicated that the protein is largely associated. The low concentration limiting s value is approximately 1.3 S, indicating a highly asymmetric monomer. In contrast, a nonmyristoylated form of the protein shows no evidence of oligomerization by velocity sedimentation and has an s value corresponding to the smallest component of NAP-22, but without the presence of higher oligomers. Sedimentation equilibrium runs indicate that there is a rapidly reversible equilibrium between monomeric and oligomeric forms of the protein followed by a slower, more irreversible association into larger aggregates. In situ atomic force microscopy of the protein deposited on mica from freshly prepared dilute solution revealed dimers on the mica surface. The values of the association constants obtained from the sedimentation equilibrium data suggest that the weight concentration of the monomer exceeds that of the dimer below a total protein concentration of 0.04 mg/ml. Since the concentration of NAP-22 in the neurons of the developing brain is approximately 0.6 mg/ml, if the protein were in solution, it would be in oligomeric form and bind specifically to cholesterol-rich domains. We demonstrate, using fluorescence resonance energy transfer, that at low concentrations, NAP-22 labeled with Texas Red binds equally well to liposomes of phosphatidylcholine either with or without the addition of 40 mol% cholesterol. Thus, oligomerization of NAP-22 contributes to its lipid selectivity during membrane binding.     

Helical structure of sugar-carrying polystyrene in aqueous solution by circular dichroism

Radical polymerization of N-p-vinylbenzyl-D-lactonamide (VLA) gave an optically active helical polymer. The stereoregularity of poly(N-p-vinylbenzyl-D-lactonamide) (PVLA) measured by 13C NMR spectroscopy showed a well-resolved sharp-line width, which was assigned to the phenyl C-1 carbon of the isotactic polystyrene (PS). The helical structure of PVLA shown by circular dichroism (CD) indicated that the aromatic groups were chirally supramolecular-packed giving optically active disaccharide units in the side chain covalently linked via an amide linkage with PS, the original PS not being optically active. The intensity of CD for PVLA (a) decreased with increasing temperature due to the change in the conformation of the phenyl group or to the breakdown of intermolecular hydrogen bonding of amide groups and (b) increased in a mixture of water and alcohol due to the increased hydrophobicity. The CD intensity for maltose-carrying PS (PVMA) was slightly higher than that of PVLA CD due to the more hydrophobic property of PVMA than PVLA.     

Three-dimensional structure of the mini-M conotoxin mr3a

Conotoxin mr3a from the venom of Conus marmoreus, a novel peptide that induces rolling seizures in mice, has the peptide sequence GCCGSFACRFGCVOCCV, where O is trans-4-hydroxyproline, and the chain is cross-linked with disulfide bonds between Cys-2 and Cys-16, Cys-3 and Cys-12, and Cys-8 and Cys-15. The tertiary structure of mr3a was determined by 2D 1H NMR in combination with a standard distance-geometry algorithm. The final set of 22 structures for the peptide had a mean global backbone RMS deviation of 0.53 +/- 0.22 A based on 51 NOE, 6 hydrogen bond, 6 phi dihedral angle, and 3 disulfide bond constraints. Conotoxin mr3a is the first example of the new mini-M branch of conopeptides in the M superfamily. Members of the maxi-M branch, whose structures are known, include the mu- and psi-conotoxins, both of which share a common disulfide bond connectivity. Although mr3a has the same arrangement of Cys residues as the mu- and psi-conotoxins, its disulfide connectivity is different. This gives mr3a a distinctive "triple-turn" backbone.     

Solution structure of mu-conotoxin GIIIA analysed by 2D-NMR and distance geometry calculations

We have investigated the structure of mu-conotoxin GIIIA by 2D-NMR methods. The assignment of 1H NMR spectra and a quantitative analysis of NOE and J-coupling data are presented. These results were used for the calculation of secondary structure elements of mu-conotoxin GIIIA. Distance geometry calculations were carried out to define the global folding of the peptide.     

Kinetics and structure during self-assembly of oppositely charged proteins in aqueous solution

Self-assembly in aqueous solution of two oppositely charged globular proteins, hen egg white lysozyme (LYS) and bovine calcium-depleted α-lactalbumin (apo α-LA), was investigated at pH 7.5. The aggregation rate of equimolar mixtures of the two proteins was determined using static and dynamic light scattering as a function of the ionic strength (15-70 mM) and protein concentration (0.28-2.8 g/L) at 25 and 45 °C. The morphology of formed supramolecular structures was observed by confocal laser scanning microscopy. When the two proteins are mixed, small aggregates were formed rapidly that subsequently grew by collision and fusion. The aggregation process led on larger length scales to irregularly shaped flocs at 25 °C, but to monodisperse homogeneous spheres at 45 °C. Both the initial rate of aggregation and the fraction of proteins that associated decreased strongly with decreasing protein concentration or increasing ionic strength but was independent of the temperature.     

The NMR structure of cyclosporin A bound to cyclophilin in aqueous solution

Cyclosporin A bound to the presumed receptor protein cyclophilin was studied in aqueous solution at pH 6.0 by nuclear magnetic resonance spectroscopy using uniform 15N- or 13C-labeling of cyclosporin A and heteronuclear spectral editing techniques. Sequence-specific assignments were obtained for all but one of the cyclosporin A proton resonances. With an input of 108 intramolecular NOEs and four vicinal 3JHN alpha coupling constants, the three-dimensional structure of cyclosporin A bound to cyclophilin was calculated with the distance geometry program DISMAN, and the structures resulting from 181 converged calculations were energy refined with the program FANTOM. A group of 120 conformers was selected on the basis of the residual constraint violations and energy criteria to represent the solution structure. The average of the pairwise root-mean-square distances calculated for the backbone atoms of the 120 structures was 0.58 A. The structure represents a novel conformation of cyclosporin A, for which the backbone conformation is significantly different from the previously reported structures in single crystals and in chloroform solution. The structure has all peptide bonds in the trans form, contains no elements of regular secondary structure and no intramolecular hydrogen bonds, and exposes nearly all polar groups to its environment. The root-mean-square distance between the backbone atoms of the crystal structure of cyclosporin A and the mean of the 120 conformers representing the NMR structure of cyclosporin A bound to cyclophilin is 2.5 A.     

Tertiary structure in N-linked oligosaccharides

Distance constraints derived from two-dimensional nuclear Overhauser effect measurements have been used to define the orientation of the Man alpha 1-3Man beta linkage in seven different N-linked oligosaccharides, all containing the common pentasaccharide core Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. Conformational invariance of the Man alpha 1-3Man beta linkage was found for those structures bearing substitutions on the Man alpha 1-3Man beta antenna. However, the presence of either a GlcNAc residue in the beta 1-4 linkage to Man beta ("bisecting GlcNAc") or a xylose residue in the beta 1-2 linkage to Man beta of the trimannosyl core was found to generate conformational transitions that were similar. These transitions were accompanied by characteristic chemical shift perturbations of proton resonances in the vicinity of the Man alpha 1-3Man beta linkage. Molecular orbital energy calculations suggest that the conformational transition between the unsubstituted and substituted cores arises from energetic constraints in the vicinity of the Man alpha 1-3Man beta linkage, rather than specific long-range interactions. These data taken together with our previous results on the Man alpha 1-6Man beta linkage [Homans, S. W., Dwek R. A., Boyd, J., Mahmoudian, M., Richards, W. G., & Rademacher, T. W. (1986) Biochemistry 25, 6342] allow us to discuss the consequences of the modulation of oligosaccharide solution conformations.     

Changes in the secondary structure of collagen after gamma-irradiation in an aqueous solution

The conformation of calf skin collagen at the secondary structure level changed after 60Co-gamma-radiation with doses ranging from 50 to 1,000 Gy in aerated aqueous solutions and in the presence of N2 and N2O. Despiralization of collagen at a dose of 1,000 Gy reached 90% in N2O, 70% in N2, and 60% in the aerated solution. The radiochemical yield of the despiralized collagen molecules was 0.007 mol/100 eV owing to OH(.). The presence of radioprotective agents upon irradiation of collagen solutions diminished considerably the injury to its secondary structure.     

NMR studies of lantibiotics. The structure of nisin in aqueous solution.

Nisin is a posttranslationally modified protein of 34 amino acids, and is a member of the class of bacteriocidal polypeptides known as lantibiotics, that contain the unusual amino acid lanthionine. Its structure in aqueous solution has been determined on the basis of NMR data, i.e. interproton distance constraints derived from nuclear Overhauser enhancement spectroscopy and torsion angle constraints derived from double-quantum-filtered correlated spectroscopy. Translation of the NMR constraints into a three-dimensional structure was carried out with the distance-geometry program DISMAN, followed by restrained energy minimization using CHARMm. The internal mobility of the peptide chain prohibited the determination of a precise overall folding of the molecule, but parts of the structure could be obtained, albeit sometimes with low resolution. The structure of nisin can best be defined as follows. The outermost N-terminal and C-terminal regions of nisin appear quite flexible, the remainder of the molecule consists of an amphiphilic N-terminal fragment (residues 3-19), joined by a flexible 'hinge' region to a rigid double-ring fragment formed by residues 23-28. The latter fragment has the appearance of a somewhat overwound alpha-helix. It is suggested, by assuming the presence of a (transient) alpha-helical structure in this part of prenisin, that the coupling between residues 23 and 26, as well as between 25 and 28, by thioether bridges, and the inversion of the C alpha chiralities at positions 23 and 25, can be rationalized.     

Tertiary structure of human complement component C5a in solution from nuclear magnetic resonance data

The tertiary structure for the region 1-63 of the 74 amino acid human complement protein C5a in solution was calculated from a large number of distance constraints derived from nuclear Overhauser effects with an angular distance geometry algorithm. The protein consists of four helices juxtaposed in an approximately antiparallel topology connected by peptide loops located at the surface of the molecule. The structures obtained for the helices are compatible with alpha-helical hydrogen-bonding patterns, which provides an explanation for the observed slow solvent exchange kinetics of the amide protons in these peptide regions. In contrast to the peptide region 1-63, no defined structure could be assigned to the C-terminal region 64-74, which increasingly acquires dynamic random coil characteristics as the end of the peptide chain is approached. An average root-mean-square deviation of 1.6 A was obtained for the alpha-carbons of the first 63 residues in the calculated ensemble of C5a structures, while the alpha-helices were determined with an average root-mean-square deviation of 0.8 A for the alpha-carbons. A comparison between the solution structure of C5a and the crystal structure of the functionally related C3a protein, as well as inferences for the interaction of C5a with its receptor on polymorphonuclear leukocytes, is discussed.     

Tertiary structure of histones]

Optical absorption and fluorescence of histones F2a and F2b were studied. An increase in pH and ionic strength induced the structure change in these histones fractions. The hydrofobic sites are formed in protein molecules and this leads to an intensification of histone-histone interactions. The change in the histone tertiary structure is of importance for processes associated with regulation of gene activity in eukaryotic cells.     

Secondary structure formations of conotoxin genes: a possible role in mediating variability

Small venomous peptides called conotoxins produced by the predatory marine snail (genus Conus) present an interesting case for mutational studies. They have a high degree of amino acid variability among them yet they possess highly conserved structural elements that are defined by cysteine residues forming disulfide bridges along the length of the mature peptide. It has been observed that codons specifying these cysteines are also highly conserved. It is unknown how such codon conservation is maintained within the mature conotoxin gene since this entire region undergoes an accelerated rate of mutation. There is evidence suggesting that nucleic acids wield some influence in mechanisms that dictate the region and frequency where mutations occur in DNA. Nucleic acids exert this effect primarily through secondary structures that bring about local peaks and troughs in the energy relief of these transient formations. Secondary structure predictions of several conotoxin genes were analyzed to see if there was any correspondence between the highly variable regions of the conotoxin. Regions of the DNA encompassing the conserved Cys codons (and several other conserved amino acid codons) have been found to correspond to predicted secondary structures of higher stabilities. In stark contrast the regions of the conotoxin that have a higher degree of variation correlate to regions of lower stability. This striking co-relation allows for a simple model of inaccessibility of a mutator to these highly conserved regions of the conotoxin gene allowing them a relative degree of resistance towards change.     

Estimation of poly A secondary structure from Raman scattering in acid aqueous solution.

In the frequency region 600–1600 cm^?1 the Raman spectra of acidic aqueous solutions of poly (rA) consist of several well-resolved lines. Four of these lines at 725, 1303, 1336 and 1508 cm^?1 demonstrate the Raman hypochromic effect of poly (rA) at pH-values of 5.73 and 5.35 as a function of the temperature.The results suggest that Raman intensity measurements are sensitive to order-disorder transitions of aqueous polynucleotides.