Conventional 1H NMR Research Articles

H and 13C Nuclear Magnetic Resonance Studies on the Stereochemical Configuration of Bis(N,N-dimethyl-2,4-dimethylglutarylamide) and Poly(N,N-dimethylacrylamide)

The conformation and configuration analysis of the dimer bis(N,N-dimethyl-2,4-dimethylglutarylamide) was carried out by means of both conventional 1H and 13C NMR spectroscopy and advanced (INEPTLR and 2D HETCOR) methods. The γ-gauche effect method appears to reproduce the observed chemical shift difference between m- and r-isomers. Stereochemical structure of poly(N,N-dimethylacrylamide) (PDMAA) was studied using proton spectrum (at the triad level) and DEPT spectra (at the triad and pentad level). For PDMAA prepared by radical polymerization, the Bernoullian statistics are required to fit the observed intensities at the pentad level.

2D NMR study of solution conformations and complete 1H and 13C chemical shifts assignments of vitamin D metabolites and analogs

In addition to conventional 1D 1H and 13C NMR, including DEPT and NOE methods, 2D -shift-correlated NMR techniques ( 1H- 1H COSY and 1H- 13C HMQC ) were used, providing an approach to the complete signal assignments for vitamin D analogs and model compounds. On the basis of NMR data, solution conformations of vitamin D and analogs were studied.

Proton NMR Studies of a Large Protein. pH, Substrate Titrations, and NOESY Experiments with Perdeuterated Yeast Phosphoglycerate Kinase Containing [ 1H]Histidine Residues

Fully deuterated yeast phosphoglycerate kinase ([ 2H]PGK) was prepared biosynthetically with only histidine side chains of normal ( 1H) isotopic composition. The 1H NMR spectrum of this enzyme([ 1H]His[ 2H]PGK) showed that the histidine side chains are clearly visible as sharp signals. Thus detailed structural studies by 1H NMR became feasible with isotope-hybrid phosphoglycerate kinase which is otherwise too large ( M r ∼ 46,000) for conventional 1H NMR studies. Proton signals of bound substrates were visible in the 1H NMR spectrum even with a substrate-to-enzyme ratio of less than 1/2 (mol/mol). The 2D NOESY spectrum of enzyme-MgdATP-glycerol 3-phosphate complex showed that, although protein concentration was very high (1.5 m M), no intraprotein cross peaks were observed other than those of intraresidue histidine NOE cross peaks. In addition, intrasubstrate NOEs and intermolecular NOEs between histidine and substrate protons were visible at a 1.5/1 substrate/enzyme (mol/mol) ratio. Paramagnetic effects of a substrate analog, Cr(III)ATP, on some of the histidine side chains indicated that the formation of the ternary enzyme-substrate complex causes large conformational changes in the enzyme.

Interaction of apocytochrome c and derived polypeptide fragments with sodium dodecyl sulfate micelles monitored by photochemically induced dynamic nuclear polarization proton NMR and fluorescence spectroscopy

The topology of apocytochrome c, the heme-free precursor of the mitochondrial protein cytochrome c, was investigated in a lipid-associated form. For this purpose photochemically induced dynamic nuclear polarization 1H nuclear magnetic resonance (CIDNP 1H NMR) spectroscopy and quenching of tryptophan and tyrosine fluorescence by acrylamide were applied to an apocytochrome c-sodium dodecyl sulfate (SDS) micellar system. A pH titration of the chemical shifts of the histidine C2 proton resonances of apocytochrome c, using conventional 1H NMR, yielded pK(a)'s of 5.9 +/- 0.1 and 6.2 +/- 0.1, which were assigned to histidine-18 and -33 and histidine-26, respectively. In the presence of SDS micelles an average pK(a) of 8.1 +/- 0.1 was obtained for all histidine C2 protons. Photo-CIDNP enhancements of the histidine, tryptophan, and tyrosine residues, contained in the intact apocytochrome c and in chemically and enzymatically prepared fragments of the precursor, were reduced in the presence of SDS micelles. Similarly, the quenching of the tryptophan fluorescence of the polypeptides by acrylamide was diminished in the presence of SDS. These results indicate the aromatic residues studied are localized in the interface of the SDS micelle.

2D COSY 1H NMR: a new tool for studying in situ brain metabolism in the living animal

2D COSY 1H NMR with surface coil has been used to resolve and assign cerebral metabolites which had previously been detected but could not be resolved or assigned in situ in the living animal by conventional ID 1H NMR. A wide range of cerebral metabolites, including alanine, N-acetyl aspartate, aspartate, choline derivatives, creatine/phosphocreatine pool, GABA, glucose, glutamate/glutamine pool, inositol, lactate and taurine were simultaneously resolved and assigned in situ in the whole animal using the 2D COSY correlation graphs. Global irreversible ischemia caused the appearance and the disappearance of cross-peaks in the 2D COSY 1H NMR map, corresponding to increases in alanine, GABA and lactate and glucose depletion.

Biochemical characterisation of para-aminophenol-induced nephrotoxic lesions in the F344 rat.

The acute biochemical effects of the nephrotoxin p-aminophenol (PAP) were studied in detail using a combination of conventional bioanalytical and 1H-NMR spectroscopic methods. Dosing PAP (25-100 mg/kg) to male F344 rats resulted in a dose-related proximal nephropathy with consequent elevations in urinary enzymes, glucose, and urine total protein as shown by conventional methodology. 1H-NMR spectroscopy at 400 MHz of urine from PAP-treated rats also revealed a characteristic glycosuria, with concomitant amino aciduria. The increased excretion of these compounds indicates functional defects in the proximal tubule and reduced solute reabsorption efficiency. In addition, 1H-NMR urinalysis and conventional enzymatic analysis showed a dose-related lactic aciduria. Other changes detected by 1H-NMR included a dose-related reduction in the excretion of citrate (confirmed by a conventional biochemical method) and an increase in the excretion of acetate. The degree of abnormalities shown by 1H-NMR urinalysis agreed well with histopathological observations and conventional biochemical indices of nephrotoxicity. 1H-NMR urinalysis therefore serves to highlight changes in the excretion of low MW urine components not routinely studied by conventional biochemical analysis.

Interaction of epidermal growth factor with micelles monitored by photochemically induced dynamic nuclear polarization-1H NMR spectroscopy.

Photochemically induced dynamic nuclear polarization (CIDNP)-1H-NMR spectroscopy has been used to study the interaction of the protein hormone epidermal growth factor (EGF) with micelles of sodium dodecyl sulfate (SDS) and dodecylphosphorylcholine (DPC). Conventional 1H-NMR spectra show that most protein resonances remain unperturbed when micelles are added to solution, which argues that the overall protein conformation is maintained in the presence of SDS or DPC at the concentrations used. Photo-CIDNP enhancements of resonances assigned to aromatic side chains of residues at the COOH terminus and beta-sheet regions of murine EGF (i.e. Trp-49, Trp-50, and Tyr-37) are considerably reduced in the presence of micelles, while resonances of aromatic side chains of residues found elsewhere on the protein surface are mostly unaffected. This suggests that the primary interaction between murine EGF and the micelle occurs at the micelle-bulk solvent interface. The overall negatively charged surface of SDS micelles tends to induce a stronger interaction with the protein compared to the zwitterionic DPC micelles, probably due to electrostatic interactions. Cleavage of the COOH-terminal pentapeptide containing both tryptophan residues enhances the already present, but weak, interaction with Tyr-10 and attenuates it with Tyr-37. A similar interaction pattern is found with rat EGF suggesting that at least concerning these two species of EGF the interaction is somewhat specific and conserved. A simple mass-action model for protein-micelle interaction is also presented.

Conformational studies by 1H nuclear magnetic resonance of the trypsin-chymotrypsin inhibitor B-III from peanuts and its enzymatically modified derivative.

The conformation of the peanut inhibitor B-III was investigated by 1H NMR at 500 MHz. Resonances for about 30% of the residues were assigned and/or identified by conventional and two-dimensional 1H NMR measurements. The conformation of the modified inhibitor B-IIIRS, obtained by the cleavage of the peptide bonds of B-III at the first reactive site, Arg(10)-Arg(11), and second reactive site, Arg-(37)-Ser(38), was also investigated. Edman degradation was used to identify resonances of the residues adjoining the scissile sites. A comparison of the behavior of the resonances corresponding to Ala(12), Tyr(15), Phe(16), and Thr(37) in B-III and B-IIIRS indicated that the only conformational differences between the two proteins were those from the S-S loops containing the two reactive sites. From the similar behavior of Leu(7), Phe(24), His(26), Ala(29), five valine, and four threonine resonances in both proteins, it is suggested that the S-S loops that do not contain a scissile bond (the core region of the inhibitor) were rigid. Thus, no conformational change of the core region was observed upon cleavage of the reactive sites.

Two-dimensional nuclear magnetic resonance at 500 MHz: the structural elucidation of a Salmonella serogroup N polysaccharide antigen

High resolution 1H and 13C nmr spectroscopy at 500 MHz and 125 MHz was used for the structural analysis of the O-antigen of Salmonellalandau, which belongs to the Kauffmann–White serogroup N. This bacterial lipopolysaccharide was extracted from whole cells and hydrolyzed by mild acid to give lipid-free O-polysaccharide. Conventional one-dimensional 1H and 13C nmr data showed the polysaccharide to contain four monosaccharides in each repeating unit and, in addition, to carry an average of one O-acetyl group for every two repeating units. Two-dimensional nmr experiments aided the unambiguous assignment of the 1H and 13C resonances and thereby permitted the structural analysis of this polysaccharide by nmr techniques alone. The structure of the de-O-acetylated repeating unit was established as [Formula: see text][Formula: see text] through the use of methods which included homonuclear shift correlated (COSY and NOESY) experiments. The interpretation of this data was supported and simplified by consideration of firmly established 13C chemical shift assignments obtained from a heteronuclear 1H/13C shift correlated experiment. A three-dimensional model of the O-antigen obtained by semi-empirical calculations is shown to be consistent with interatomic distance constraints imposed by data from 2-D NOESY and one-dimensional nOe difference spectroscopy.

Metal Ion Binding to Parvalbumin. A Proton NMR Study.

The 1H NMR spectra of carp parvalbumin saturated with Ca2+, Cd2+, La3+ and Lu3+ were compared, using 2D 1H NMR techniques as well as conventional 1H NMR spectra. The Ca2+ and Cd2+ saturated parvalbumin (with both high affinity Ca2+-binding sites occupied) gave rise to very similar spectra. This shows that these two species have almost identical protein conformations. The 1H NMR spectrum from the Ln3+ saturated parvalbumins deviated from the other two and it was therefore concluded that Cd2+ is a better probe for Ca2+ than Ln3+ in parvalbumin and probably also for related calcium binding proteins. The addition of excess of divalent metal ions, such as Mg2+ or Ca2+, causes small changes in the chemical shift of some methyl resonances. This is presumably caused by binding of these metal ions to a third site close to the CD site which is made up of the carboxylic groups from Glu 60 and Asp 61.

NMR spectra of systems with restricted motion: Cross-linked polymer gels

Swollen cross-linked polystyrene, and a parallel solution of linear polystyrene in CCl 4, were studied by 1H NMR, both conventional and with magic angle rotation (MAR-NMR), and by 13C NMR lineshape, T 1 and NOEF. The 1H MAR-NMR spectra of the cross-linked polymer were found to be almost identical to conventional 1H NMR spectra of the linear polymer in a broad temperature range. Also, the 13C T 1 and NOE parameters were equal for the cross-linked and linear polymer indicating little difference in the dynamics of rapid internal motions. The characteristic “super-Lorentzian” lineshape of conventional 1H NMR spectra of the cross-linked polymer gel is analyzed in terms of a convolution of a residual dipolar broadening function G( ν − ν 0) with a narrow lineshape function S( ν − ν 0)defined by rapid internal motions.