Uropygiols: confirmation of structure by proton magnetic resonance

Lipids were extracted from excised uropygial glands of domestic chickens and the wax diesters were isolated by preparative thin-layer chromatography (TLC). The diesters were hydrolyzed and the liberated diols were resolved by boric acid TLC into two fractions. These were investigated by proton magnetic resonance at 360 MHz of the free diols and of their acetonide derivatives. The results showed that the cis and trans acetonides, formed from the erythro and threo isomers of the diols, respectively, could be distinguished by the degree of magnetic nonequivalence of the two acetonide methyl groups in each molecule. On the presumption that the cis isomer should show the greater nonequivalence of the methyl groups, this configuration was assigned to the acetonides of these diols which had the lesser TLC mobility on boric acid/silica gel. This agrees with the assignment of configuration made by earlier workers on the basis of the relative TLC mobility of the diol isomers on boric acid/silica gel, but was contrary to a previous assignment based on gas-liquid chromatographic (GLC) retention times. We conclude that the erythro isomers of the diols are characterized by lower mobility on boric acid TLC, as well as on silica gel TLC, and form acetonides that have longer retention times on GLC, and greater nonequivalence of the acetonide methyl groups in the NMR spectrum, than do the acetonides of the threo isomers.     

Solution and ion-complexed conformations of beauvericin determined by proton magnetic resonance spectroscopy

The conformational properties of the cyclohexadepsipeptide antibiotic Beauvericin have been investigated by 1H-NMR spectroscopy in polar (C2H3O2H) and non-polar (CCl4, C62H6, C2HCl3) solvents and in two solvent mixtures; one a mixture of a polar and non-polar solvent (C2H3O2H/CCl4) and the other an aromatic solvent in a non-polar environment (C62H6/CCl4). The ion-complexation properties of Beauvericin with alkali metal halides (Li+, Na+, K+, Cs+) have also been studied. It is demonstrated that changes in chemical shifts of Beauvericin with concentration, with polarity of solvent or with added alkali metal ion reflect changes not only in the solvent properties but also changes in backbone conformation and changes due to ion-complexation, where appropriate, and therefore cannot be used, by themselves, to determine the conformation of the molecule, its self-aggregation properties, or the stoichiometry of the metal ion-complex. The backbone conformations of Beauvericin in different environments are determined by methods that are independent of chemical shift analysis; i.e., by measurements of 5J(HH) magnitudes observed between the alpha-CH protons of the L-phenylalanine and D-hydroxyisovaleric acid (DHyIv) residues and by nuclear Overhauser effect measurements observed between alpha-CH(HyIv) and (N)-CH3(Phe) proton signals. In the knowledge of these results the chemical shifts of Beauvericin in different environments can then be rationalised. It is found that the conformation of Beauvericin in a polar solvent is different from that found in a non-polar solvent and from that found for the in the ion-complexed form is similar to that found in non-polar solvents. By taking into account the conformational properties of the L-phenylalanine and DHyIv side-chains, it is possible to assign unambiguously the magnetically non-equivalent beta-CH2(Phe) and gamma Me(HyIv) proton signals and so elucidate the complete conformational behaviour of the uncomplexed forms of Beauvericin in a polar and a non-polar environment, and of the ion-complexed form of Beauvericin in a polar solvent.     

Three-dimensional structure of an alpha-amylase inhibitor HAIM as determined by nuclear magnetic resonance methods

The three-dimensional structure of an alpha-amylase inhibitor, HAIM, composed of 78 amino acids, was analyzed by two-dimensional NMR techniques. Sequence-specific assignments were made for the amino acid residues from Ile-6 to Cys-72. Distance geometry analysis of the interresidue NOEs revealed that the HAIM molecule consists of two beta-sheets, as is the case in a homologous alpha-amylase inhibitor, Tendamistat, though one of its beta-strands is much shorter than that of Tendamistat. The combination of molecular modeling from Tendamistat and distance geometry analysis was confirmed to be useful for our purpose.     

Elucidation of glycolipid structure by proton nuclear magnetic resonance spectroscopy

The primary structure of the oligosaccharide moiety of a glycosphingolipid can be elucidated by employing high-field proton nuclear magnetic resonance (NMR) spectroscopy. Information with respect to the composition and configuration of its sugar residues, and the sequence and linkage sites of the oligosaccharide chain can be obtained by employing a variety of one- and two-dimensional techniques. The latter include both scalar and dipolar correlated two-dimensional NMR spectroscopy. These techniques are also useful in establishing the solution conformation (secondary structure) of the oligosaccharide moiety. Examples in utilizing these techniques in elucidating the primary and secondary structures of glycolipids are presented.     

Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution.

The 1:1 complex of the mutant Antp(C39----S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three-dimensional structure were based on the use of the fully 15N-labelled protein, two-dimensional homonuclear proton NOESY with 15N(omega 2) half-filter, and heteronuclear three-dimensional NMR experiments. Based on nearly complete sequence-specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H-1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix-turn-helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix-turn-helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39----S) homeodomain no defined spatial structure could be found for the apparently flexible N-terminal segment comprising residues 0-6.     

Self-association of puromycin as studied by proton magnetic resonance spectroscopy

The self-association of puromycin has been studied using proton magnetic resonance spectroscopy. The concentration, temperature and pH dependence studies of the proton chemical shifts of the adenine protons indicate that puromycin in aqueous solution at pD 7.4 self associates predominantly through adenine-adenine interaction. At this pD, the amino group of the aminoacyl segment of puromycin has been demonstrated to exist in a equilibrium blend of protonated and non-protonated forms. At pD 2.6, PM is found to exist predominantly in the monomeric from in which the methyl groups of the 6N-dimethyladenine are found to be non-equivalent due to hindered rotation about the C6-N6 bond.     

The structure of two alanine containing ferrichromes: Sequence determination by proton magnetic resonance

Summary Metal coordination confers an extraordinary structural stability to the ferrichromes which, independent of their variable amino acid composition, results in a basically unperturbed conformation for all the homologous peptides in the series. The proton magnetic resonance (pmr) characteristics for Al³⁺ analogues (alumichromes) reflect this conformational isomorphism in usual solvents so that single site substitutions are clearly recognized in the pmr spectra. Thus, the substitution of glycine byl-alanine orl-serine introduce new resonances characteristic of the sidechains and alter the pattern of the amide NH pmr region in that doublets substitute for glycyl triplets at the same site. Since for glycine- andl-serine-containing alumichromes the resonances have already been identified, it is possible to unequivocally establish the primary structure of the twol-alanyl homologues ferrichrome C ( ) and sake colorant A ( ) on the basis of the comparative pmr spectra of their Al³⁺ analogues, namely, alumichrome C and alumisake. The resonance assignment, and hence the site occupancy, is substantiated by the temperature coefficients of the NH chemical shifts, rates of¹H-²H exchange and homonuclear proton spin decoupling experiments centered on the NH spectral region. Occupancy of site 1 by a glycine residue is observed for all known ferrichromes, which serves to conserve a hairpin turn. This method of obtaining sequence information should prove of general use for other systems of homologous polypeptides, provided their conformations are not affected by the residue substitutions.     

Solution structure of ascidian trypsin inhibitor determined by nuclear magnetic resonance spectroscopy

The three-dimensional solution structure of ascidian trypsin inhibitor (ATI), a 55 amino acid residue protein with four disulfide bridges, was determined by means of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy. The resulting structure of ATI was characterized by an alpha-helical conformation in residues 35-42 and a three-stranded antiparallel beta-sheet in residues 22-26, 29-32, and 48-50. The presence of an alpha-helical conformation was predicted from the consensus sequences of the cystine-stabilized alpha-helical (CSH) motif, which is characterized by an alpha-helix structure in the Cys-X(1)-X(2)-X(3)-Cys portion (corresponding to residues 37-41), linking to the Cys-X-Cys portion (corresponding to residues 12-14) folded in an extended structure. The secondary structure and the overall folding of the main chain of ATI were very similar to those of the Kazal-type inhibitors, such as Japanese quail ovomucoid third domain (OMJPQ3) and leech-derived tryptase inhibitor form C (LDTI-C), although ATI does not show extensive sequence homology to these inhibitors except for a few amino acid residues and six of eight half-cystines. On the basis of these findings, we realign the amino acid sequences of representative Kazal-type inhibitors including ATI and discuss the unique structure of ATI with four disulfide bridges.     

Solution structure of apamin determined by nuclear magnetic resonance and distance geometry

The solution structure of the bee venom neurotoxin apamin has been determined with a distance geometry program using distance constraints derived from NMR. Twenty embedded structures were generated and refined by using the program DSPACE. After error minimization using both conjugate gradient and dynamics algorithms, six structures had very low residual error. Comparisons of these show that the backbone of the peptide is quite well-defined with the largest rms difference between backbone atoms in these structures of 1.34 A. The side chains have far fewer constraints and show greater variability in their positions. The structure derived here is generally consistent with the qualitative model previously described, with most differences occurring in the loop between the beta-turn (residues 2-5) and the C-terminal alpha-helix (residues 9-17). Comparisons are made with previously derived models from NMR data and other methods.     

Linoleate-rich acylglucosylceramides of pig epidermis: structure determination by proton magnetic resonance

The structure of the linoleate-rich acylglycosylceramides isolated from pig epidermis has been reinvestigated. Gas-liquid chromatographic analysis of the alditol acetates produced from the sugar component indicated that 90% of the hexose is glucose while the remaining 10% is galactose. The predominance of the beta-D-glucosyl group was confirmed by 360 MHz proton magnetic resonance spectroscopy. The magnetic resonance method was also used to prove that the ester-linked linoleic acid is actually attached to the omega-hydroxyl group of the long chain hydroxyacid, not to the sugar as had been reported previously. A key spectral feature supporting this new structural assignment was a triplet at 3.82 ppm, which indicates methylene protons between another methylene and an ester linkage. After saponification, this signal moved to 3.33 ppm, a chemical shift expected for a methylene bearing a free hydroxyl group. Furthermore, all of the sugar ring protons could be accounted for both before and after acetylation. No evidence was found to suggest that an ester is attached to the sugar ring in the native material. It is concluded that the principal porcine epidermal acylglycosylceramide is 1-beta-D-glucosyl-N-(omega-O-linoleoyl)-triacontanoylsphingosine.