Oxygen-17 n.m.r. of peptides

Peptide-17O chemical shifts of linear dipeptides with and without protecting groups in H2O, CH3OH, CH2Cl2, CHCl3, CCl4, CH3CN and DMSO were between 256-350 ppm downfield from external water. Increasing solvent H-bond donating ability correlated with shifts to higher field. The 17O resonance of several cyclic dipeptides appeared at higher field relative to comparable linear dipeptides (303-317 p.p.m. vs. 327-337 p.p.m.). Separate signals were simultaneously observed by 13C and 17O n.m.r. for cis and trans N-tert.-butyl-formamide in binary mixtures with H2O, (CH3)2CO, and CCl4. The differences in the 17O nuclear screening of the amide isomers and most probably for cis and trans peptides were independent of contributions from H-bonding at the amide or peptide linkage, apparently reflecting differences between geometric isomers in electron distribution and through space effects. Peptide-17O of Gly-Ala, Gly-Leu and Gly-Glu in aqueous solution experienced upfield shifts of 6-12 p.p.m. and 12-16 p.p.m. upon deprotonation of the C-terminal COOH and of the N-terminal NH3+ groups respectively. These observations were rationalized in terms of the attendant changes in substituent effects, especially on the pi electron donating ability of the N atom at the peptide linkage and increased partial negative charge on the peptide oxygen. Temperature studies of peptide-17O of Gly-Ala between pH 1.5-9.0 revealed a chemical shift coefficient of 0.08 p.p.m./degree K and similar behavior of T1 and T2 relaxation times. Ea for molecular rotation was 5 kcal/mol between 301-331 degrees K. Rotational correlation times, tau c, were within the range expected from the Stokes-Einstein relation.     

Proton n.m.r. of ferredoxin from Clostridium pasteurianum

Proton magnetic resonance spectra at 500 MHz are reported for the oxidized and reduced forms of the 2[4Fe-4S]-ferredoxin from Clostridium pasteurianum. The reduced protein showed additional peaks in the 10–60 ppm region, which were previously unobserved, and there were significant differences between oxidized and reduced states in the whole region. The electron exchange rate in partially reduced ferredoxin is slow on the n.m.r. time scale when reduced with sodium dithionite, but fast when zinc reduced methyl viologen is used as reducing agent. We explain the difference between fast and slow exchange as being due to the different chemical properties of the two reducing agents.     

13C n.m.r. study of L-alanine peptides

The di-, tri-, and tetrapeptides of L-alanine have been studied in aqueous solution by 13C n.m.r. spectroscopy at 25 and 50 MHz. By using selectively 13C enriched analogs containing either 90% 13C methyl or carbonyl carbons and measurements as a function of pH, assignment of the chemical shifts of the peptides has been made. T1 and NOE measurements of the peptides in their cationic, anionic, and zwitterionic states have been recorded as a function of concentration. The results show considerable segmental motion along the backbone carbons of the peptides, with only small changes occurring in the dynamic motions of the peptides as their charge states are altered. The lack of concentration dependence of the chemical shift and T1 values, as well as the similarity of T1 values for individual peptides in the three charge states, indicate that the peptides do not self-associate in aqueous solution.     

Proton n.m.r. of ferrodoxin from Clostridium pasteurianum

Proton magnetic resonance spectra at 500 MHz are reported for the oxidized and reduced forms of the 2[4Fe-4S]-ferredoxin from Clostridium pasteurianum. The reduced protein showed additional peaks in the 10–60 ppm region, which were previously unobserved, and there were significant differences between oxidized and reduced states in the whole region. The electron exchange rate in partially reduced ferredoxin is slow on the n.m.r. time scale when reduced with sodium dithionite, but fast when zinc reduced methyl viologen is used as reducing agent. We explain the difference between fast and slow exchange as being due to the different chemical properties of the two reducing agents.     

The interaction of sugars with borate: an n.m.r. spectroscopic study

¹¹B n.m.r. spectroscopy studies of solutions of sugars in the presence of borax have shown the existence of boron-containing complexes. Chemical-shift values indicate the size of the ring in which boron is involved. Use of this method and p.m.r. spectroscopy showed that D-glucose forms a 1,2-furanoid and 1,2-pyranoid complex in the presence of borax and benzeneboronic acid.     

15N n.m.r. spectroscopy: 36. 1H n.m.r. and 15N n.m.r.spectroscopic investigation on the protonation of polypeptides

¹⁵N n.m.r. (9.12 MHz) spectra of acetamide, polyglycine, poly([l-alanine) and poly(l-leucine) were measured in various acidic solvents. These solvents include dichloroacetic acid (DCA), trifluoroacetic acid (TFA), methane sulphonic acid (MSA) and fluorosulphonic acid (FSA). Full protonation of both amides and polypeptides causes downfield shifts of 17–20 ppm. Furthermore, the concentration dependence of the chemical shift was measured. In solvents which cause partial protonation, decreasing concentration of amide groups may cause downfield shifts up to 8.5 ppm, while in the case of full protonation or in the absence of protonation no concentration dependence is observable. The protonation of peptide groups induces H/D-exchange of the αC proton which was monitored by ¹H n.m.r. spectroscopy. The mechanism of this H/D-exchange is discussed.     

Progress in n.m.r. zeugmatography imaging

Applications of nuclear magnetic resonance (n.m.r.) zeugmatographic imaging to medical diagnosis and to medical, physiological, and biological research require the development of appropriate imaging instrumentation and ancillary techniques, as well as an understanding of the biological significance of the imaging results. A whole body imaging system, relying primarily upon reconstruction from projections, is under development in the expectation that the reconstruction approach will be the most practical one for many purposes. In addition, injectable magnetic reagents that can selectively change tissue water relaxation times and image contrast are under development so as to increase the specificity and versatility of the measurements. If very high magnetic fields are employed, 31P n.m.r. zeugmatography may be practical at very low resolution for human diagnostic studies and for experiments on perfused organs and small animals. Preliminary images, showing the spatial distributions of different phosphorus metabolites in the compartments of test objects, have been obtained at 146 MHz by reconstruction techniques.     

Experimental protocol for tissue discrimination in vitro by n.m.r.

We have examined the n.m.r. relaxation times T1 and T2 of water protons for liver (mouse, human) and brain (mouse) at different temperatures and subjected to various conditions of conservation and degeneration. After tissue degeneration, T1 and T2 behave differently and their variations are characteristic of each tissue type. The results show that the initial values at +4 degrees C are consistent when the experimental protocol formulated in this study is followed.     

Conformational characteristics of receptor-selective opioid peptides. 1H n.m.r. and c.d. spectroscopic studies of delta-kephalin and [Val4]morphiceptin.

An investigation on the conformations of highly receptor-selective opioid peptides was carried out to gain further understanding of the structure-activity relationship of endogenous enkephalins. The preferred conformations of a highly mu-selective [Val4]morphiceptin and a highly delta-selective delta-kephalin have been probed by 1H n.m.r. solvent-, concentration- and temperature-dependences of amide protons to take the folded conformations stabilized by an intramolecular hydrogen bond and the anti-parallely extended dimeric structures respectively. Their possible stereo-conformations were proposed, based on the analyses of the vicinal coupling constants (JHNC alpha H). The conformational difference between the mu- and delta-selective opioid peptides was further ascertained by the c.d. measurements. The c.d. spectra of the mu-selective peptides show negative bands in the range of 210-230 nm, while those of the delta-selective ones show the opposite positive bands. A correlation between c.d. spectra and receptor-selectivity was possible.     

The preferred conformations of protected homodito homoheptamethionine peptides. A1 H n.m.r. study in deuterochloroform medium

Detailed analyses of the conformations of the homo-oligopeptide series, Boc-(L-Met)n-OME n = 2--7, in deuterochloroform have been carried out with proton n.m.r. and IR spectroscopy. Well-resolved high field n.m.r. spectra with assignments for the NH and alpha-CH resonances of these homo-methionine peptides are presented. Extensive n.m.r. concentration-dependent chemical shift studies are combined with IR results to delineate the involvement of the various methionine NH protons in intra- and/or intermolecular hydrogen bonding. N.m.r. chemical shift dependencies with temperature and solvent, DMSO-d6, are used to explore the strength of the hydrogen bonds for the various oligopeptides. At low concentrations, where peptide aggregation is absent, the dipeptide is found to be disordered. The tetra- to heptapeptides possess intramolecular hydrogen bonded seven-membered rings at internal residues. The number of internal rings and the oligopeptide self-association increase with increasing peptide chainlength. At intermediate concentrations associations of peptide molecules with folded structures occur with initial association at the C-terminal region. At high concentrations, "in-register" associated extended beta structures are formed.     

Conformations of proline-containing cyclic peptides. 1H and 13C n.m.r. evidence for a solvent stabilized All-Cis X-Pro conformer of Cyclo-(Pro-Gly-Gly-Pro)2

As inferred from 13C, 1H n.m.r. data, CD measurements and ion-binding experiments, the title molecule can assume two major C2 symmetric conformations. One of these has an all-trans X-Pro peptide backbone and two 1 comes from 4 intramolecular H-bonds and represents the predominant (greater than or equal to 95%) form in D2O and nonpolar (CD3CN) solvents. Stabilized by specific solvent-solute interactions, the other conformer becomes competitive (45%) in DMSO solution. It is shown to possess a four-cis X-Pro skeleton and no intramolecular H-bonds. The Mg++ complex of the cyclic peptide in CD3CN is again C2 symmetric and its formation proceeds via a slow trans leads to cis isomerization of two X-Pro peptide bonds.     

1H n.m.r. study of geletin gels

Proton spin-lattice and spin-spin relaxation times have been measured in the gelatin-water system during gelatin at various concentrations and quenching temperatures. The spin-spin relaxation time is shown to be a good parameter for following the kinetics of gelation and can be related to the amount of helices measured by polarimetry. The results are analysed within the framework of the Zimmerman-Brittin theory.     

Measurement of intracellular potassium ion concentrations by n.m.r

39K n.m.r. was used to detect and quantify K+ within human erythrocytes. A shift reagent consisting of an anionic complex of dysprosium(III) with sodium tripolyphosphate permitted a distinction to be made between K+ inside and outside erythrocytes. Intracellular K+ concentrations determined by this method were similar to values obtained by flame photometry.     

Some biophysical applications of motional contrast in n.m.r. microscopy

The principal advantage of the n.m.r. imaging method lies in the specific contrasts which are available. In this work we describe the use of velocity and diffusion contrast methods in biophysical applications and at microscopic spatial resolution. In the first example, involving water-protein interactions, the relationship between water self-diffusion and water concentration, as measured using pulsed gradient spin echo n.m.r., is shown. It is demonstrated that this relationship can be used to provide a water concentration image. The result is compared with the conventional proton density and transverse relaxation maps. The next example concerns the use of dynamic n.m.r. microscopy to obtain water diffusion and velocity maps for wheat grain in vivo. Finally we suggest how the method may be used in the study of polymer-water interactions in an unusual adjunct to conventional polymer self-diffusion studies.     

Conformational analysis of 3,4-epoxytetrahydropyran by n.m.r. spectroscopy

The conformational equilibrium of the title compound has been determined by correlating its n.m.r. parameters with those of its 2,2,6,6-tetradeuterio derivative and trans- and cis-2-tert-butyl-4,5-epoxytetrahydropyran. The high preference (ΔG ～ ?0.8 kcal/mol) for the half-chair conformation in which the pyranoid oxygen is furthest from the oxirane oxygen atom can be interpreted in terms of electrostatic interactions between the two oxygen atoms.     

Polypeptide fluoroacetate derivatives. Synthesis and 19F n.m.r

The synthesis and characterization of monofluoroacetyl (MFAc) functionalized haptens are described. These were covalently bound to polypeptide carriers (bovine serum albumin and poly-D-lysine) by primary amine/succinimide ester or primary amine/acid chloride coupling. Epitopic densities of the resulting antigens were determined by both 19F n.m.r. and picryl sulfonic acid assays. 19F n.m.r. experiments defining the stability of MFAc ester and amide linkages as a function of media (including in vitro), time, and temperature are presented. These results indicate that MFAc functionalized antigens are well suited for further immunologic studies.