1H-, 13C- and 31P-NMR studies of dioctanoylphosphatidylcholine and dioctanoylthiophosphatidylcholine

Coupling constants and chemical shifts were measured for dioctanoylphosphatidylcholine and its thio analogue in a CDCl3/CD3OD solvent mixture. Replacing the bridging oxygen atom of the CH-CH2-O-P portion of the phosphatidylcholine molecule with a sulfur atom affects chemical shifts and coupling constants in the glycerol backbone portion of the molecule as well as in the choline head group region. Preferred conformations about selected bonds in the phospholipids were determined from the vicinal 1H-1H, 31P-1H and 31P-13C coupling constants. A reduction of the 31P T2* (effective spin-spin relaxation time) for the thio analogue, as well as changes in the relative chemical shifts of 13C nuclei in the acyl chains, suggest a somewhat greater degree of aggregation for the thio analogue. The quadrupolar coupling constant 1J(14N-13C) for the choline methyls of either analogue, however, indicates that aggregation of these phospholipids in the CDCl3/CD3OD solvent mixture is not significant. Differences in conformation between dioctanoylphosphatidylcholine and its thio analogue may be responsible for their differences in chemical and physical properties.     

1H- and 31P-NMR studies on smooth muscle of bullfrog stomach

³¹P-NMR spectra of bullfrog stomach smooth muscle showed peaks for creatine phosphate (4.8 μmol·g⁻¹ wet wt.), ATP (3.6), inorganic phosphate (P_i, 2.4), phosphomonoesters (3.0) and phosphodiesters (3.3). The intracellular pH was 7.3, and calculated from the chemical shift of P_i. ¹H-NMR spectra of smooth muscle yielded peaks of 2.9 for lactate, 6.6 for total creatine (creatine phosphate + creatine) and methyl protons of choline tentatively assigned to glycerolphosphorylcholine or to membrane phospholipids. Creatine phosphate and ATP decreased under anaerobic conditions, and intracellular acidification was observed with the concomitant increase in lactate. ³¹P saturation transfer studies showed that saturation of the γ-ATP resonance reduced the intensity of creatine phosphate to 60% of its control value, and the measured T₁ value of creatine phosphate was 2.4 s with saturation. The calculated forward flux of the creatine kinase reaction (decomposition direction of creatine phosphate) was 0.77 μmol·g⁻¹ wet wt.·s⁻¹. The creatine kinase flux was approx. 100-times larger than the ATP turnover rate, calculated from the oxygen consumption rate with the assumption, P/O = 3. In conclusion, the creatine kinase reaction is at equilibrium in resting smooth muscle of bullfrog stomach.     

Hairpins in a DNA site for topoisomerase II studied by 1H- and 31P-NMR.

1H- and 31P-NMR and UV-absorption studies were carried out with the oligonucleotide strands d(AGCT-TATC-ATC-GATAAGCT) (-ATC-) and d(AGCTTATC-GAT-GATAAGCT) (-GAT-) contained in the strongest and salt resistant cleavage site for topoisomerase II in pBR322 DNA. We found that the two oligonucleotides were stabilized under a hairpin structure characterized by a eight base pair stem and a three base loop at low DNA and salt concentrations. In such experimental conditions, only the -GAT- oligonucleotide displayed a partial homoduplex structure in slow equilibrium with its folded structure. Temperature dependencies of imino protons showed that the partial homoduplex of -GAT- melted at a lower temperature than the hairpin structure. It was suggested that the appearance of the partial homoduplex in -GAT- is related to the formation of two stabilizing (G.T) mismatched base pairs in the central loop of this structure. Finally, it was inferred from the dispersion of chemical shifts in the 31P-NMR spectra that the distortions affecting the backbone of the hairpin loop are larger in the case of -ATC- compared with -GAT-. At the same time NOEs proved that the base stacking was stronger within the loop of the -ATC- hairpin.     

31P-NMR assignment and conformational study of NADPH bound to Lactobacillus casei dihydrofolate reductase based on two-dimensional 1H-31P-heteronuclear and 1H-detected 1H-31P-shift-correlation experiments.

For any detailed NMR conformational study of a protein-ligand complex it is essential to have specific resonance assignments. We have now assigned the pyrophosphate 31P resonances in spectra of NADPH bound to Lactobacillus casei dihydrofolate reductase (DHFR) by using a combination of 1H-31P-heteronuclear shift-correlation (HETCOR), 1H-31P-heteronuclear multiple-quantum-coherence correlation spectroscopy (HMQC-COSY), 1H-1H COSY, homonuclear Hartmann-Hahn (HOHAHA) and NOE spectroscopy (NOESY) experiments. The nicotinamide pyrophosphate phosphorus, P(n), has been unequivocally assigned to a signal (-14.07 ppm) which shows a large 3JP-O-C-H coupling constant. Such a coupling constant when combined with the appropriate Karplus relationship provides conformational information about the P-O-C-H torsion angle. The torsion angle changes by 65 degrees +/- 10 degrees for the binary complex compared with the value in free NADPH. The observed coupling constants for the binary (DHFR--NADPH) and ternary (DHFR--NADPH--methotrexate) complexes (12.3 and 10.5 +/- 0.6 Hz, respectively) indicate that the pyrophosphate group has a similar conformation in the two complexes.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

31P-NMR saturation transfer study of the in vivo kinetics of arginine kinase in Carcinus crab leg muscle

The kinetics of the reaction catalyzed by arginine kinase have been determined at 9.5 and 23°C for in vivo leg muscle of Carcinus maenas (the common shore crab) using the noninvasive technique of ³¹P-NMR spectroscopy. Concentrations of mobile phosphorus metabolites were the same at both temperatures: 78.7 mM for arginine phosphate, 9.0 mM for adenosine triphosphate (ATP), and 2.6 mM for inorganic phosphate (P_i), as estimated from NMR resonance intensities and literature values for ATP concentration as assayed by traditional biochemical methods. Apparent unidirectional rate constants for formation of ATP from arginine phosphate and ADP were 0.09 s^?1 at 9.5°C and 0.27 s^?1 at 23°C. Pseudo-first-order rate constants for arginine phosphate generation from Arg and ATP were 0.38 and 1.10 s^?1 at 9.5 and 23°C, respectively. In vivo Q₁₀ for the arginine kinase reaction between 9.5 and 23°C was thus 2.2 for both directions. When the kinetic data are analyzed using the Arrhenius equation, activation energies of 126 kJ/mol for ATP formation and 105 kJ/mol for arginine phosphate formation are found. The measured chemical fluxes through arginine kinase in the forward reaction (arginine phosphate hydrolysis) were twice those in the reverse reaction, consistent with either compartmentation of substrates or participation of substrates in alternative metabolic pathways.     

Determination of free creatine and phosphocreatine concentrations in the isolated perfused rat heart by 1H- and 31P-NMR.

To measure free creatine in the isolated perfused rat heart, the concentration of phosphocreatine, and phosphocreatine plus creatine (sigma Cr) were measured by 31P- and 1H-NMR, respectively. Quantification was performed in the presence and absence of an intraventricular balloon filled with a known amount of PCr, which acted as an external standard. Total (free plus bound) phosphocreatine and creatine were measured by HPLC analysis of extracts from the same hearts, freeze-clamped at the end of the perfusions. A greater concentration of creatine (mumol/g dry wt.) in the perfused rat heart was measured by HPLC analysis (40.3 +/- 2.38 (11)) as compared to NMR (34.6 +/- 1.95 (11)), whilst no significant difference was observed in the measurement of phosphocreatine between the two assay methods. Consequently, a greater sigma Cr was measured by HPLC. This work suggests that the majority of Cr in the heart is NMR visible and unbound, so available to interact with creatine kinase. The lower free ADP concentration calculated from NMR measurements (53.3 +/- 3.80 microM (9)) was not significantly different from that determined by HPLC analysis (56.9 +/- 5.90 microM (9)). This suggests that the concentration of free ADP in the heart is higher than values where it can regulate oxidative phosphorylation most effectively.     

31P and 1H NMR studies of the structure of enzyme-bound substrate complexes of lobster muscle arginine kinase: relaxation measurements with Mn(II) and Co(II)

The paramagnetic effects of Mn(II) and Co(II) on the spin-lattice relaxation rates of 31P nuclei of ATP and ADP and of Mn(II) on the spin-lattice relaxation rate of the delta protons of arginine bound to arginine kinase from lobster tail muscle have been measured. Temperature variation of 31P relaxation rates in E.MnADP and E.MnATP yields activation energies (delta E) in the range 6-10 kcal/mol. Thus, the 31P relaxation rates in these complexes are exchange limited and cannot provide structural information. However, the relaxation rates in E.CoADP and E.CoATP exhibit frequency dependence and delta E values in the range 1-2 kcal/mol; i.e., these rates depend upon 31P-Co(II) distances. These distances were calculated to be in the range 3.2-4.5 A, appropriate for direct coordination between Co(II) and the phosphoryl groups. The paramagnetic effect of Mn(II) on the 1H spin-lattice relaxation rate of the delta protons of arginine in the E.MnADP.Arg complex was also measured at three frequencies (viz., 200, 300, and 470 MHz). These 1H experiments were performed in the presence of sufficient excess of arginine to be observable over the protein background but with MnADP exclusively in the enzyme-bound form so that the enhancement in the relaxation rates of the delta protons of arginine arises entirely from the enzyme-bound complex. Both the observed frequency dependence of these rates and the delta E less than or equal to 1.0 +/- 0.3 kcal/mol indicate that this rate depends on the 1H-Mn(II) distances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation and dynamic structure of poly(inosinic acid) in neutral aqueous solution by 1H-, 2H-, 13C-, and 31P-NMR spectroscopy

The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: ¹H (90 and 250 MHz), ²H (13.8 MHz), ¹³C (75.4 MHz), and ³¹P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the ¹H and ¹³C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T₁ ratios between the sugar protons, T₁ (H₁′)/T₁ (H₃′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T₂. For a given temperature between 22 and 62°C, the ¹³C-{¹H} NOE value is practically the same for seven protonated carbons (C₂, C₈, C_1′, C_2′, C_3′, C_4′, C_5′). This is also true for the T₁ ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τ_c, for all protons and carbons. The τ_c values determined from either the ¹³C-{¹H} NOE or proton T₁ ratios, T₁(90 MHz)/T₁(250 MHz), and/or deuterium transverse relaxation time T₂ agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T₁ (³¹P) and ³¹P-{¹H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data.     

Comparative 2H- and 31P-NMR study on the properties of palmitoyllysophosphatidylcholine in bilayers with gramicidin, cholesterol and dipalmitoylphosphatidylcholine

The stoichiometric palmitoyllysophosphatidylcholine (lysoPC)/gramicidin (4:1, mol/mol) lamellar complex (Killian, J.A., De Kruijff, B., Van Echteld, C.J.A., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1983) Biochim. Biophys. Acta 728, 141-144) is a useful model system to investigate the various aspects of lipid protein interactions. To study the effect of gramicidin on local order and motion of 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC) we employed 31P and 2H nuclear magnetic resonance (NMR) using selectively deuterated lysoPC's and we compared the results to those obtained for lysoPC in bilayers with cholesterol (1:1, mol/mol) and dipalmitoylphosphatidylcholine (DPPC) (1:4, mol/mol). 2H-NMR experiments on acyl chain deuterated lysoPC showed similar quadrupole splittings in the liquid crystalline state for the lysoPC/DPPC and the lysoPC/gramicidin samples. In the lysoPC/cholesterol sample an increase of the quadrupole splitting was found. T1 measurements showed that gramicidin decreases the lysoPC acyl chain motion, especially at the C12 position. In the lysoPC/cholesterol sample an increase of motion was observed as compared to lysoPC in fluid bilayers of DPPC. 31P-NMR and 2-H-NMR measurements of lysoPC, deuterated at the alpha- and beta-position of the choline moiety, indicated an increase in headgroup flexibility in all samples as compared to the parent compound DPPC. In addition, a change in headgroup conformation was observed. The alpha- and beta-segments in all samples exhibited concerted motion. It was found that also in the polar headgroup gramicidin induces a decrease of the rate of motion.     

Mapping of glucose and glucose-6-phosphate binding sites on bovine brain hexokinase. A 1H- and 31P-NMR investigation

Inhibition of bovine brain hexokinase by its product, glucose 6-phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6-phosphate on the enzyme with respect to a divalent-cation-binding site characterized previously [Jarori, G. K., Kasturi, S. R. & Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211, 258-268]. The paramagnetic effect of enzyme-bound Mn(II) on the spin-lattice relaxation rates (T-1(1] of ligand nuclei (1H and 31P) in E.Mn(II).Glc and E.Mn(II).Glc6P complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1-H alpha, C1-H beta and C2-H beta of glucose in the E.Mn(II).Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5-30 degrees C at 500 MHz. The ligand nuclear relaxation rates in E.Mn(II).Glc are field-dependent and the Arrhenius plot yields an activation energy (delta E) of 16.7-20.9 kJ/mol. Similar measurements have also been carried out on C1-H alpha, C1-H beta and C6-31P at 270 MHz (1H) and 202.5 MHz (31P) for the E.Mn(II).Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5-30 degrees C, which yielded delta E = 9.2 kJ/mol. The electron-nuclear dipolar correlation time (tau c), determined from the field-dependent measurements of proton relaxation rates in the E.Mn(II).Glc complex, is 0.22-1.27 ns. The distances determined between Mn(II) and C1-H of glucose and glucose 6-phosphate are approximately 1.1 nm and approximately 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare, U. W. (1988) J. Biol. Chem. 263, 15492-15498], suggest that glucose and glucose 6-phosphate may bind to very nearly the same region of the enzyme. The structure of the binary Glc6P.Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co-ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of approximately 0.5-0.6 nm from the cation.     

NMR studies of ligand carboxylate group interactions with arginine residues in complexes of Lactobacillus casei dihydrofolate reductase with substrates and substrate analogues

In a series of complexes of Lactobacillus casei dihydrofolate reductase (DHFR) formed with substrates and substrate analogues, the (1)H/(15)N NMR chemical shifts for the guanidino group of the conserved Arg 57 residue were found to be sensitive to the mode of binding of their H(eta) protons to the charged oxygen atoms in ligand carboxylate groups. In all cases, Arg 57 showed four nonequivalent H(eta) signals indicating hindered rotation about the N(epsilon)-C(zeta) and C(zeta)-N(eta) bonds. The H(eta)(12) and H(eta)(22) protons have large downfield shifts as expected for a symmetrical end-on interaction with the ligand carboxylate group. The chemical shifts are essentially the same in the complexes with folate and p-aminobenzoyl-L-glutamate (PABG) and similar to those found previously for the methotrexate complex reflecting the strong and similar hydrogen bonds formed with the carboxylate oxygens. Interestingly, the rates of rotation about the N(epsilon)-C(zeta) bond for the complexes containing the weakly binding PABG fragment are almost identical to those measured in the complex with methotrexate, which binds 10(7) times more tightly. In the methotrexate complex, this rotation depends on correlated rotations about the N(epsilon)-C(zeta) bond of Arg 57 and the C(alpha)-C' bond of the ligand glutamate alpha-carboxylate group. Thus, even in a fragment such as PABG, which has a much faster off-rate, the carboxylate group binds to the enzyme in a similar way to that in a parent molecule such as folate and methotrexate with the rotation about the N(epsilon)-C(zeta) bond of Arg 57 being essentially the same in all the different complexes.     

Conformationally-restricted arginine analogues as alternative substrates and inhibitors of nitric oxide synthases.

Conformationally restricted arginine analogues (1-5) were synthesized and found to be alternative substrates or inhibitors of the three isozymes of nitric oxide synthase (NOS). A comparison of k(cat)/Km values shows that (E)-3,4-didehydro-D,L-arginine (1) is a much better substrate than the corresponding (Z)-isomer (2) and 3-guanidino-D,L-phenylglycine (3), although none is as good a substrate as is arginine; 5-keto-D,L-arginine (4) is not a substrate, but is an inhibitor of the three isozymes. Therefore, it appears that arginine binds to all of the NOS isozymes in an extended (E-like) conformation. None of the compounds exhibits time-dependent inhibition of NOS, but they are competitive reversible inhibitors. Based on the earlier report that N(omega)-propyl-L-arginine is a highly selective nNOS inhibitor (Zhang, H. Q.; Fast, W.; Marletta, M.; Martasek, P.; Silverman, R. B. J. Med. Chem. 1997, 40, 3869), (E)-N(omega)-propyl-3,4-didehydro-D,L-arginine (5) was synthesized, but it was shown to be weakly potent and only a mildly selective inhibitor of NOS. Imposing conformational rigidity on an arginine backbone does not appear to be a favorable approach for selective NOS inhibition.     

31P NMR studies of the arginine kinase reaction. Equilibrium constants and exchange rates at stoichiometric enzyme concentration.

The arginine kinase reaction, the reversible transfer of the terminal phosphoryl group of ATP to L-arginine, has been investigated by the technique of 31P NMR at catalytic and stoichiometric concentrations of the enzyme. Three of the four substrates, ATP, ADP, and P-arginine produce easily distinguishable resonances in the 31P NMR spectrum, thus permitting a determination of equilibrium constants from the integrated areas of the resonances. From the linewidths, the exchange rates between reactants and products may be evaluated. At pH 7.25 and a temperature of 12 degrees, the equilibrium constant at catalytic enzyme concentration: Keq = [MgADP] [P-arginine]/[MgATP] [L-arginine], is found to be 0.10 +/- 0.02 and that at stoichiometric enzyme concentration: K'eq = [E-MgADP] [E-P-arginine]/[E-MgATP] [E-arginine] to be 1.56 +/- 0.5. Thus, as the enzyme concentration increased, the production of P-arginine is increasingly favored. From the NMR line shapes in the presence of excess enzyme, the rate of the single step, the transfer of the phosphoryl group on the surface of the enzyme is found to be 192 +/- 15 s-1 in the forward direction, i.e. from E-MgATP, and 154 +/- 15 s-1 in the reverse direction from E-P-argine. At 12 degrees and pH 7.25, the rate of the overall reaction in the forward direction was determined from kinetic measurements to be 19 s-1, an order of magnitude slower than the rate measured by NMR. It can, therefore, be concluded that the interconversion of substrates on the surface of the enzyme is not the rate-determining step in the overal reaction. From the equilibrium constants and other known data the dissociation constant of P-arginine from its enzyme complex can be determined and is found to be 100 muM.     

1H-NMR and (31)P-NMR analysis of energy metabolism of quiescent and motile turbot (Psetta maxima) spermatozoa

31P-NMR and (1)H-NMR were used to monitor changes of several compounds with high-energy bonds and metabolites prior to and after the initiation of motility of turbot spermatozoa (Psetta maxima). The obtained (31)P-NMR spectra revealed the presence of phosphomonoesters, phosphodiester, intracellular inorganic phosphate (Pi), phosphocreatine (PCr), and free nucleotide triphosphate. Following the activation of motility, the di- and tri-phosphate nucleotides, PCr, phosphomonoesters levels dropped while Pi levels increased. A significant increase of lactate was also seen at the end of the swimming phase. The compositions of seminal fluid and urine were also determined. Lipoproteins, formic acid, amino acid, and citric acid were detected in seminal fluid. Dimethyl amine, trimethylamine, and trimethylamine oxyde were found in urine. These data suggest that at least a part of the energy required during the swimming phase results from anaerobic fermentation and oxidative phosphorylation. J. Exp. Zool. 286:513-522, 2000.     

Bisintercalation of ditercalinium into a d(CpGpApTpCpG)2 minihelix: a 1H- and 31P-NMR study

The structure of the complex formed in aqueous solution between ditercalinium, a bisintercalating drug, and the self-complementary hexanucleotide d(CpGpApTpCpG)2 is investigated by 400-MHz 1H-nmr and 162-MHz 31P-nmr. Whatever the drug to helix ratio, ditercalinium occurred in the bound form, whereas free and complexed hexanucleotide are in slow exchange. This allows unambiguous resonance assignment through two-dimensional chemical exchange experiments. The strong upfield shifts measured on most aromatic protons on both drug and bases as well as on DNA imino protons are consistent with bisintercalation of the dimer. Nuclear Overhauser effects observed between drug and nucleotide protons give a defined geometry for complexation, and suggest a DNA conformational change upon drug binding.     

Influence of stigmastanol and stigmastanyl-phosphorylcholine, two plasma cholesterol lowering substances, on synthetic phospholipid membranes. A 2H- and 31P-NMR study.

Cholesterol, stigmastanol, and stigmastanyl-phosphorylcholine (ST-PC) were incorporated into model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). POPC and ST-PC were deuterated at the lipid headgroup, DOPC at the cis-double bonds. The influence of the three sterols on the motion and conformation of the lipid headgroups and the hydrocarbon chains was monitored with 2H- and 31P-NMR. All three sterols were freely miscible with the lipid matrix in concentrations of up to 50 mol% without inducing phase separations or nonbilayer structures. However, the molecules exert quite different effects on the phospholipid bilayer. Cholesterol and stigmastanol are largely buried in the hydrocarbon part of the membrane, distinctly restricting the flexing motions of the fatty acyl chains whereas the conformation of the phospholipid headgroups is little affected. In contrast, ST-PC is anchored with its headgroup in the layer of phospholipid dipoles, preventing an extensive penetration of the sterol ring into the hydrocarbon layer. Hence ST-PC has almost no effect on the hydrocarbon chains but induces a characteristic conformational change of the phospholipid headgroups. The 2H- and 31P-NMR spectra of mixed phospholipid/ST-PC membranes further demonstrate that the PC headgroup of ST-PC has a similar orientation as the surrounding phosphatidylcholine headgroups. For both types of molecules the -P-N+ dipole is essentially parallel to the membrane surface. Addition of ST-PC induces a small rotation of the POPC headgroup towards the water phase.