13C NMR studies on [4-13C] cholesterol incorporated in sonicated phosphatidylcholine vesicles

1. 90.5 MHz 13C NMR linewidth measurements were performed on mixed sonicated [4-13C] cholesterol/phosphatidylcholine vesicles of different fatty acid composition. 2. From the Dy3+ -induced shift of the C4 resonance of cholesterol it suggested that this part of the molecule is localized in the ester bond region of the bilayer. 3. The local motion of the cholesterol ring system is restricted and independent of fatty acid composition. 4. At cholesterol concentrations below 30 mol percent the ring system becomes more immobilised when the fatty acids of the phosphatidylcholine molecules enter the gel state.     

Conformational analysis of morphiceptin by NMR spectroscopy

Three exorphins, beta-casomorphin-5, morphiceptin and its D-Pro4 analog, were studied in DMSO by means of 1H and 13C NMR spectroscopy, with the aim of detecting conformational features of potential biological significance for the mu opioid activity since the presence of two Pro residues restricts the accessible conformational space more than in all other peptides. It is found that the conformational mixtures present in solution contain relevant fractions of folded conformers, a feature that assures the observation of four different Tyr OH signals in the 500 MHz spectrum of morphiceptin. The conformer distribution of (very active) (D-Pro4)-morphiceptin is different from those of its (less active) congeners.     

Conformational transitions and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state 13C NMR

Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high-resolution solid-state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local alpha-helical form is present around Gly10 whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a local beta-sheet form as viewed from Gly10 and Phe22, and both beta-sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from alpha-helix to beta-sheet, and from random coil to beta-sheet forms occurred in the central and C-terminus regions, respectively, during the fibril formation. The increased 13C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two-step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local beta-sheet conformation at the central region and exhibited a random coil at the C-terminus region. Not only a hydrophobic interaction among the amphiphilic alpha-helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel beta-sheets at pH 7.5 and a mixture of antiparallel and parallel beta-sheets at pH 3.3.     

13C NMR spectral analysis of eperuane diterpenes

The ¹³C NMR spectra of some eperuane diterpenes have been recorded and the signals assigned. The substituent shielding effects in these compounds, in comparison with those observed in other series of diterpenes, are also presented.     

NMR study of rapid water diffusion across lipid bilayers in dipalmitoyl lecithin vesicles

A method for measurement of rapid diffusional exchange between external and internal water in lecithin vesicles is described. Paramagnetic ions were inserted inside DPL vesicles and the NMR relaxation times for water protons were measured as a function of temperature. It was found that water diffusion rate is described by a single activation energy of 15±1 kcal/mole in the temperature range 16 – 35°C and exhibits a maximum at 44°C. The permeability of DPL vesicles to water was calculated to 16–18 × 10^?4 cm/s at 44°C and 1.7 × 10^?4 cm/s at 20°C.     

13C NMR analysis of methionine sulfoxide in protein.

The 13C epsilon NMR signal of methionine sulfoxide is 22.6 ppm downfield from that of methionine. This affords a method by which the extent of methionine oxidation can be determined in intact protein. We demonstrate the utility of this approach with beta-galactosidase enriched with 13C in its methionine methyls.     

Structure analysis of proximadiol (cryptomeridiol) by 13C NMR spectroscopy

The sesquiterpene diol with antispasmodic properties, earlier isolated from Cymbopogon proximus, is shown to be identical with cryptomeridiol.     

Conformational exchange on the microsecond time scale in alpha-helix and beta-hairpin peptides measured by 13C NMR transverse relaxation

13C-NMR relaxation experiments (T(1), T(2), T(1)(rho), and NOE) were performed on selectively enriched residues in two peptides, one hydrophobic staple alpha-helix-forming peptide GFSKAELAKARAAKRGGY and one beta-hairpin-forming peptide RGITVNGKTYGR, in water and in water/trifluoroethanol (TFE). Exchange contributions, R(ex), to spin-spin relaxation rates for (13)C(alpha) and (13)C(beta) groups were derived and were ascribed to be mainly due to peptide folding-unfolding. To evaluate the exchange time, tau(ex), from R(ex), the chemical shift difference between folded and unfolded states, Deltadelta, and the populations of these states, p(i), were determined from the temperature dependence of (13)C chemical shifts. For both peptides, values for tau(ex) fell in the 1 micros to 10 micros range. Under conditions where the peptides are most folded (water/TFE, 5 degrees C), tau(ex) values for all residues in each respective peptide were essentially the same, supporting the presence of a global folding-unfolding exchange process. Rounded-up average tau(ex) values were 4 micros for the helix peptide and 9 micros for the hairpin peptide. This 2-3-fold difference in exchange times between helix and hairpin peptides is consistent with that observed for folding-unfolding of other small peptides.     

A 31P NMR study of the thermal transition of dipalmitoyl lecithin vesicles.

Phosphorous-31 nuclear magnetic resonance (31P NMR) was used to study the liquid crystalline transition of sonicated dipalmitoyl lecithin vesicles in D2O. Linewidths were dependent upon temperature and changed dramatically through the transition region. The potential usefulness of 31P NMR spectroscopy for probing the thermal behavior of phospholipid membranes is evaluated and discussed.     

A 1H and 13C NMR study of motional changes of dipalmitoyl lecithin associated with the pretransition

Motional changes of the dipalmitoyl lecithin molecule associated with the pretransition in multibilayers are investigated by proton-enhanced 13C-NMR and proton spin-locking experiments. The nitrogen-bound methyl groups of the polar head exhibit faster motion and more disorder in the intermediate phase compared with the gel phase. Although little or no change occurs in the hydrocarbon chain order at the pretransition, the corresponding motional correlation time changes by one order of magnitude. This is consistent with a model involving rotational motion of the hydrocarbon chains about their long axes: in the gel phase the motion is such that neighboring chains make an oscillating disrotatory motion, while in contrast, in the intermediate phase a quasi-free chain rotation takes place. Earlier contradicting results of Davies, J., 1979, Biophys. J., 27:339-358, and ourselves, Trahms, L., and E. Boroske, 1979, Biochim, Biophys. Acta. 552:189-193, are explained by this model.     

A 13C NMR spin-lattice relaxation study of the interaction of myelin proteins with lipid vesicles

Natural abundance 13C nuclear magnetic spin-lattice relaxation times have been measured for bovine brain phosphatidylserine vesicles with and without bound proteins. The relaxation times were lower than published values for the corresponding nuclei in egg phosphatidylcholine, but showed the same trend, with relaxation times increasing along the acyl chains away from the polar headgroup. These times were inversely related to the degree of saturation of the lipid. Cytochrome c caused insignificant changes in the lipid acyl chain relaxation rates but reduced the resonance intensities, in agreement with Brown and Wüthrich (Biochem. Biophys. Acta 468 (1977) 389). In contrast, the basic protein from bovine myelin did not affect the intensities but reduced the relaxation times for 13C nuclei near the bilayer centre, and for nuclei near carbon-carbon double bonds. These proteins also dramatically broadened the serine headgroup carboxyl resonance. It appears, in accord with other recent evidence, that the basic protein does penetrate the hydrophobic region of the bilayer (possibly to the centre), producing quantitatively similar changes in the relaxation rates to proteolipid protein, an integral membrane protein.     

Derivatives of C-branched monosaccharides studied by 13C NMR

13C NMR spectra of some 3-C branched D-allofuranoses and D-ribofuranoses were obtained and interpreted. The impact of attaching the alkyl substitute to the monosaccharides on chemical shifting of the adjacent carbon atoms was shown. The experimental data are useful for elucidating structures of analogous compounds by 13C NMR.     

Interaction of two prolamins with 1-13C oleic acid by 13C NMR

In this paper, we analyzed the interaction of Z19 prolamin from a BR451 maize variety and pennisetin from a BRS1501 pearl millet variety with 1-(13)C-enriched oleic acid (OA) by (13)C NMR in solution. In both proteins, we identified the presence of free fatty acids by NMR in solid state and solution. The interactions were analyzed at the protein/OA molar ratios of 1:1 and 1:4. In the Z19/OA 1:1 mixture in 70% ethanol and 30% D(2)O, the chemical shift of OA C1 was 182.9 ppm, about 3 ppm above that of the pure OA in the same solvent. In contrast, upon addition of OA to the pennisetin (1:1), the chemical-shift value slightly decreased by less than 1 ppm. The chemical-shift titration curve of OA C1 in an apparent pH range of 5.5-7.3 shifted by approximately 0.3 pH units toward higher pH values in the pennisetin/OA 1:1 complex relative to the pure OA. The results obtained for the pennisetin/OA 1:4 mixture were similar to the complexes at a 1:1 molar ratio. A significant difference was observed between the 1:1 and 1:4 curves for Z19. The titration curve for Z19/OA 1:1 suggested specific binding at the sites with electrostatic interaction.     

The lateral diffusion coefficient of lecithin molecules in single bilayer vesicles studied by 14N NMR

The ¹⁴N nuclear relaxation times T₁ and T₂ in egg yolk phosphatidylcholine have been observed in single bilayer vesicles dispersed in the media of different viscosities, ¹H₂O and ²H₂O. The lateral diffusion coefficient of lipid molecule D has been calculated according to the method reported earlier: D = 2.2 × 10^?8cm²s^?1 in ¹H₂O and 2.1 × 10^?8cm²s^?1 in ²H₂O at 20°C. They are in excellent agreement. This result gives a strong basis of usefulness of ¹⁴N NMR method in the evaluation of D without introducing any system perturbation.     

13C NMR isotopomer analysis of anaplerotic pathways in INS-1 cells

Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic beta-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was approximately 40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mm stimulated insulin secretion approximately 4-fold, and was associated with a approximately 4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux approximately 6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in beta-cells and that anaplerosis through GDH does not play a major role in this process.     

13C-NMR Investigation of the insertion of the bee venom melittin into lecithin vesicles

The orientation of melittin in lecithin membranes was investigated by means of ¹³C-NMR spectroscopy. Phospholipase-free melittin was labeled with ¹³C-methyl groups at the -amino side chains of lysine 7, 21, and 23. From the pH dependence of the corresponding ¹³C resonances, pK values of the lysine residues were derived that were different for free and membrane-bound melittin. The shift reagent Pr(NO₃)₃ induced shifts in the ¹³C resonance position of all three lysines when melittin and the shift reagent were added to a lecithin vesicle suspension, whereas Pr³⁺ ions included in the inner volume of the vesicles did not affect the ¹³C resonances of melittin bound to the outer vesicle membrane. A wedge-like structure was derived for the membrane-bound melittin, the lysine side chains of which are freely accessible to the aqueous solvent.Abbreviation NOE Nuclear Overhauser Enhancement     

Solid-state 13C NMR spectroscopy of a 13C carbonyl-labeled polypeptide

High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For ¹³C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled ¹⁴N from the peptide bond. Here the ¹³C₁-Gly₂ site in gramicidin A is studied both as a dry powder and in a fully hydrated lipid bilayer environment. Linewidths reported for the oriented samples are a factor of five narrower than those reported elsewhere, and previous misinterpretations of the linewidths are corrected. The observed frequency from oriented samples is shown to be consistent with the recently determined structure for this site in the gramicidin backbone. It is also shown that, whereas a dipolar coupling between ¹³C and ¹⁴N is apparent in dry preparations of the polypeptide, in a hydrated bilayer the dipolar coupling is absent, presumably due to a `self-decoupling' mechanism.     

Metabolism of [2-13C]succinate in renal cells determined by 13C NMR

[2-13C]Succinate has been used to examine the metabolic carbon flux from the Krebs cycle in rat renal proximal convoluted tubular (PCT) cells under physiological and pathophysiological conditions. Therefore, we developed a mathematical model that enabled us to determine the metabolic fluxes of the Krebs cycle. A mathematical model for the calculation of flux from [2-13C]succinate was used to determine fluxes in rat PCT cells during chronic acidosis in the presence and absence of 0.1 mM angiotensin II. The relative carbon efflux via glutamate dehydrogenase in rat renal PCT cells increases during chronic acidosis from 0.27 to 0.39, whereas this carbon flux is not affected by the presence of peptide hormone angiotensin II in the incubation medium. The fraction of intermediate 13C-labelled oxaloacetate transformed into the phosphoenolpyruvate and aspartate pools increases significantly from 0.41 to 0.57 in the case of chronic acidosis. The carbon efflux is not affected by angiotensin II. The 13C-NMR data also show that the carbon efflux through phosphoenolpyruvate carboxykinase increases from 0.35 to 0.56 in rat renal PCT cells derived from chronic acidotic animals, as well as in the presence of angiotensin II. The present results indicate that angiotensin II affects only the flux through phosphoenolcarboxykinase, whereas chronic acidosis increases the flux through phosphoenolpyruvate carboxykinase as well as the gluconeogenic flux.