Conformation and motion of the choline head group in bilayers of dipalmitoyl-3-sn-phosphatidylcholine.

The conformation and motion of the choline head group in lipid bilayers above and below the gel-to-liquid crystal transition point are studied by means of deuterium and phosphorus magnetic resonance. For this purpose dipalmitoyl-3-sn-phosphatidylcholine is selectively deuterated at various positions on the choline and glycerol constituents. The residual deuteron quadrupole couplings and the phosphorus chemical-shift anisotropy of the corresponding lipid-water mixtures yield quantitative information on the segmental motions. The choline methyl group is only slightly hindered in its movement, but the motional freedom becomes increasingly restricted the closer the segment is located to the glycerol backbone. The average value of the OC-CN bond rotation angle changes with temperature. Increasing the temperature rotates the choline methyl group into the vicinity of the phosphorus atom. The choline group as a whole is thus characterized by a flexible, temperature-dependent structure. Its orientation in space is not fixed, either parallel or perpendicular to the bilayer surface. Instead all segments execute angular oscillations with varying degrees of restriction around the normal on the bilayer surface. The gel-to-liquid crystal phase transition at 41 degrees is clearly reflected in the deuterium and phosphorus resonance spectra of the choline moiety, while no change is observed at 34 degrees. The calorimetric pretransition at 34 degrees seems not to be associated with a conformational change in the choline group.     

Deuterium order parameters in relation to thermodynamic properties of a phospholiped bilayer. A statistical mechanical interpretation.

The physical properties of bilayers of dipalmitoyl-3-sn-phosphatidylcholine are analyzed in terms of a statistical model proposed by Marcelja (S. Marcelja (1974), Biochim. Biophys. Acta 367, 165). The model is used to calculate the segmental order parameters of the hydrocarbon chains, the transition temperature of the crystalline leads to liquid crystalline phase transition, the entropy change of the transition, the bilayer thickness, and the linear thermal expansion coefficient. The theoretical predictions are in excellent agreement with experimental results obtained by deuterium magnetic resonance, differential scanning calorimetry, and X-ray diffraction. The model yields the probabilities of trans and gauche conformations and also those of more specific conformational defects like kinks or jogs.     

Response of the headgroup of phosphatidylglycerol to membrane surface charge as studied by deuterium and phosphorus-31 nuclear magnetic resonance

The response to membrane surface charge of the glycerol headgroup of dimyristoyl-phosphatidylglycerol (DMPG) was investigated via deuterium and phosphorus-31 nuclear magnetic resonance spectroscopy. The membrane surface charge was manipulated by adding various amounts of neutral dimyristoylphosphatidylcholine (DMPC) and/or positively charged didodecyldimethylammonium bromide (DDAB) to the negatively charged DMPG, selectively deuterated at the alpha and beta segments of its glycerol headgroup. The deuterium and phosphorus-31 nuclear magnetic resonance spectra were all characteristic of random dispersions of liquid-crystalline lipids in a bilayer configuration. Differential scanning calorimetry showed that all mixtures investigated exhibited gel to liquid-crystalline phase transitions below 35 degrees C. Measurements of the deuterium quadrupole splitting and of the phosphorus-31 chemical shift anisotropy lead to the following observations. (1) Dilution of the negative surface charge density by the addition of DMPC had little effect on the quadrupole splitting from either alpha- or beta-deuterated DMPG. (2) Direct cancellation of the negative surface charge density by addition of DDAB led to a progressive decrease in the quadrupole splitting measured from alpha-deuterated DMPG, while the quadrupole splitting measured from beta-deuterated DMPG increased. For alpha-deuterated DMPG addition of 0.3 mole fraction of DDAB resulted in the appearance of two distinct quadrupole splittings. No such effect was observed for beta-deuterated DMPG.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

Determination of conformational properties of glycolipid head groups by 2H NMR of oriented multibilayers

The conformations and orientations of the glucose and glycerol moieties of a monoglucosyl lipid in hydrated bilayers have been determined in detail by deuterium nuclear magnetic resonance (2H NMR). Multibilayer membranes of 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)glycerol (DTGL), of dimyristoylphosphatidylcholine (DMPC), and of a mixture of DTGL and DMPC were oriented between glass plates. The glucolipid was selectively labeled with deuterium on the pyranose ring and at C3 of glycerol, whereas DMPC was labeled at the C4 position of the sn-2 chain. Quadrupolar splittings were measured as a function of the angle between the bilayer normal and the magnetic field direction. The results establish that the director of motional averaging, the direction about which motion and order are axially symmetric, is the bilayer normal for all the head group, the glycerol backbone, and the hydrophobic core. Segmental order parameters were determined to be 0.45, 0.65, and 0.40, respectively, for the various regions of DTGL in the membranes. The latter results indicate that there is some motion on the time scale of 10(5) s-1 about the C1'(glucose)-O-C3(glycerol) glycosidic bond but that its amplitude is very restricted. Comparison of 1H-decoupled and 1H-coupled 2H NMR spectra of the C3-labeled glycolipid gave estimates of the 2H-2H dipolar coupling between the deuterons at this position. The orientation of the glycerol C3 hydroxymethylene subunit was calculated relative to the bilayer normal, and the C2-C3 bond was found to be tilted away from the bilayer normal by 3 +/- 1 degree.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein mobility and self-association by deuterium nuclear magnetic resonance.

Hen egg white lysozyme has been prepared in which the C epsilon position of the single histidine residue is substituted by a deuterium atom as a nondisturbing stable isotope probe. The deuterium nuclear magnetic resonance (2H NMR) spectrum in H2O shows a broad resonance (500--1000 Hz) due to the histidine deuteron and a sharp signal from residual HOD. The line width of the deuterium signal increases with pH, reflecting the self-association of lysozyme which is known to involve this histidine [shindo, H., Cohen, J.S., & Rupley, J. A. (1977) Biochemistry 16, 3879]. Correlation times calculated from spin-spin relaxation times (T2) derived from the 2H widths indicate that His-15 is restricted in motion and that lysozyme is predominantly dimerized at pH 7.5. Controls carried out with [epsilon-2H]imidazole showed a small pH dependence of the spin-lattice relaxation time (T1), which parallels the 2H chemical shift change upon ionization of the imidazole. Similar results cannot generally be observed by proton nuclear magnetic resonance (1H NMR) because of paramagnetic relaxation due to trace metal ion impurities. The pH dependence of the 2H T1 values indicates a change in the 2H quadrupole coupling constant upon protonation of the imidazole ring.     

Quantitative determination of the conformation of cyclic 3',5'-adenosine monophosphate in solution using lanthanide ions as nuclear magnetic resonance probes

The conformation of cyclic 3′,5′-adenosine monophosphate in deuterium oxide has been determined at pH 2.0 and pH 5.5, using lanthanide ions as paramagnetic nuclear magnetic resonance probes.The lanthanide ion-induced shifts in the nuclear magnetic resonance energy for a given nucleus are dependent on the geometric position of that nucleus relative to the bound lanthanide ion. As expected, these shifts are pseudocontact in origin and are consistent with axial symmetry. Analysis of the concentration dependence of the shift shows that the lanthanide ion is bound to the phosphate entity giving a 1:1 complex. Further, base stacking and other intermolecular interactions are negligible.To confirm the conformation, which is found from a computer search with the above shift data, we have measured the changes in relaxation times, T₁ and T₂, induced by binding of Gd³⁺. The geometric dependence of these relaxation effects is different from that of shifts, being dependent only on distance. The agreement of these data with the computer “shift” conformation is satisfactory.Some ³¹P nuclear magnetic resonance experiments were done to confirm the metal co-ordination position although, here, there are contact contributions to both shift and relaxation.The computer program finds the conformations that have the correct geometry to account for the shift data, by searching all possible conformations. Non-bond rotations were used as a method of changing the pucker of the phosphate and ribose rings, the position of the base being defined by a single bond rotation. The nuclear magnetic resonance data and minimum van der Waals' distances were used as “active filters” in the computer search.At both values of the pH we have found closely related families of solutions, with the pucker of the phosphate and ribose rings roughly similar to those in an approximate X-ray study of cyclic AMP. The orientation of the base varies with pH.     

Carbon-13 magnetic resonance of carboxymethylated human carbonic anhydrase B. Chemical shift and spin--lattice relaxation studies.

We have previously prepared Ntau-carbosymethylhistidine-200 human carbonic anhydrase B using 90% [1-13C]bromoacetate and have observed the 13C NMR resonance of the enriched carboxylate now covalently attached in the active site. We report here chemical shift studies of the zinc-free carboxymethylated enzyme and its Co2+-substituted form, as well as relaxation studies of the resonance in the zinc enzyme at three frequencies (15.04, 25.15, and 90.5 MHz). The chemical shift and relaxation data are both consistent with the immobilization of the carboxylate at pH 8 and its approach or coordination to the zinc. The relaxation data indicate that lowering the pH to 5.5 leads to internal motion of the carboxymethyl moiety, consistent with the chemical shift evidence for the disruption of the proposed zinc--carboxylate coordination. Inhibitor binding at either pH 5.5 or 8.0 eliminates whatever internal motion might be present. The relaxation data have been interpreted using theoretical calculations on dipolar and chemical shift anisotropy contributions. The combined results indicate that the catalytic consequences of the carboxymethylation may be due to the proposed zinc--carboxylate coordination and need not result from the disruption of any role that histidine-200 might play in the catalytic mechanism.     

Partitioning behaviour of 1-hexanol into lipid membranes as studied by deuterium NMR spectroscopy

Deuterium nuclear magnetic resonance (NMR) spectroscopy was used to study the partitioning behaviour of 1-hexanol specifically deuterated in the alpha-position into model lipid bilayers. In all systems studied, the observed deuterium NMR lineshapes were time-dependent. Initially, 1-hexanol-d2 gave rise to an isotropic deuterium resonance with a different chemical shift from that of aqueous 1-hexanol-d2. After equilibration over a period of days, a broader spectral component characteristic of a spherically-averaged powder-pattern was observed. The quadrupole anisotropy of the 1-hexanol-d2 giving rise to the broad spectrum depended upon the cholesterol content of the membrane. From quantitation of the anisotropic to isotropic deuterium NMR spectra, the partition coefficients of 1-hexanol-d2 in a number of bilayer systems (asolectin and phosphatidylcholine bilayers (the latter with and without cholesterol] were determined. The partitioning of 1-hexanol-d2 into red blood cell membranes, and a suspension of lipids extracted from red blood cell membranes, was also examined. It is suggested that 1-hexanol, and probably other lipophiles, can partition to either the bilayer surface or the bilayer interior in a time-dependent manner.     

Orientational order and rotational diffusion of the head group in the bilayer membrane. A nuclear magnetic resonance study.

An order parameter-based interpretation is applied to the temperature dependence of the deuterium magnetic resonance splittings and the anisotropic contribution to the chemical shift for 31P from the head groups of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). It is shown that the rotational motion of the molecule about its long axis is not a free rotational motion as normally assumed, but instead a biased one. Changes in the degree of biasing appear to be primarily responsible for the variation of the NMR spectra with temperature. The degree of biasing is described by orientational order parameters. With the use of these order parameters, it is shown that the temperature dependence of the anisotropic contribution to the chemical shift for 31P can be predicted from that of the deuterium quadrupole splittings.     

Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group

The binding of the charged form of two local anesthetics, dibucaine and etidocaine, to bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was measured simultaneously with ultraviolet spectroscopy and deuterium magnetic resonance. Because of their amphiphilic molecular structure, both drugs intercalate between the lipid molecules, increasing the surface area and imparting a positive electric charge onto the membrane. The ultraviolet (UV) binding isotherms were therefore analyzed in terms of a model which specifically took into account the bilayer expansion as well as the charge-induced concentration variations near the membrane surface. By formulating a quantitative expression for the change in surface area upon drug intercalation and combining it with the Gouy-Chapman theory, the binding of charged dibucaine and etidocaine to the lipid membrane was best described by a partition equilibrium, with surface partition coefficients of 660 +/- 80 M-1 and 11 +/- 2 M-1 for dibucaine and etidocaine, respectively (pH 5.5, 0.1 M NaCl/50 mM buffer). Deuterium magnetic resonance demonstrated further that the binding of drug changed the head-group conformation of the lipid molecules. Invoking the intercalation model, a linear variation of the deuterium quadrupole splittings of the choline segments with the surface charge density was observed, suggesting that the phosphocholine head-group may act as a 'molecular electrometer' with respect to surface charges.     

Interaction of charged and uncharged calcium channel antagonists with phospholipid membranes. Binding equilibrium, binding enthalpy, and membrane location.

The membrane location and the binding mechanism of two Ca2+ channel antagonists, amlodipine and nimodipine, in pure lipid membranes were investigated with deuterium and phosphorus-31 nuclear magnetic resonance, with thermodynamic methods such as high-sensitivity titration calorimetry, and by measuring the membrane surface charge via the zeta-potential. The two drugs exhibit quite different physical-chemical properties. The noncharged nimodipine is strongly hydrophobic, and selective deuteration of the lipid membrane reveals a homogeneous distribution of nimodipine across the whole hydrocarbon layer, but no interaction at the lipid headgroup level. The membrane behavior of the amiphiphilic amlodipine (electric charge z = +1) is distinctly more complex. Deuterium magnetic resonance demonstrates that amlodipine adopts a well-defined position in the bilayer membrane. In particular, the charged ethanolamine side group of amlodipine is located near the water-lipid interface, interacting with the dipoles of the headgroup region according to a nonspecific, electrostatic mechanism and inducing a reorientation of the phosphocholine dipoles toward the water phase. At the level of the hydrocarbon segment, the nonpolar ring system of amlodipine interacts specifically with the cis double bond of the membrane lipid, forming a weak association complex. With increasing amlodipine concentration the deuterium signal of the cis double bond gradually loses intensity, a phenomenon previously observed only in related studies on protein-lipid interactions. The binding equilibrium of amlodipine to phosphatidylcholine membranes was studied by measuring the electrophoretic mobility of lipid vesicles and with a centrifugation assay. Hydrophobic interactions of the nonpolar ring systems and electrostatic repulsions at the membrane surface contribute to the binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational changes of the phosphatidylcholine headgroup due to membrane dehydration. A 2H-NMR study

The influence of hydration on the orientation of the phosphocholine dipole in bilayer membranes was studied with nuclear magnetic resonance. The phosphocholine headgroup of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was deuterated at the two methylene segments. Phosphorus-31 and deuterium nuclear magnetic resonance measurements were made as a function of hydration in the range of 10-70 wt.% H2O revealing a distinct change in the alignment of the phosphocholine headgroup. With decreasing hydration the N+ end of the phosphocholine head group dipole moves closer to the hydrocarbon layer. The conformational change induced by the loss of water molecules at the membrane surface is qualitatively similar to that observed upon addition of polyhydroxyl compounds.     

NMR studies of oxyleghemoglobin: assignment of distal histidine proton resonances and evidence for pH-dependent changes in conformation

The resonance of the C-2 proton of the distal histidine has been assigned in the 400 MHz 1H-NMR spectrum of soybean ozyleghemoglobin a. This resonance is subject to a very large ring current shift from the heme and occurs to high field of the residual HO2H peak. The pH dependence was measured from a series of nuclear Overhauser effect difference spectra over a range of pH values. The resonance moves to high field with decreasing pH and reflects titration of a one proton-dissociable group with pK 5.5. Resonances of the heme substituents and distal amino acid side-chains are also sensitive to this titration. Changes in ring-current shifts and nuclear Overhauser effects indicate that a conformational change occurs in the heme pocket upon titration of the pK 5.5 group. We propose that protonation of the distal histidine with pK 5.5 is accompanied by movement of the imidazole ring towards the heme normal. This movement would allow interaction between the ligated oxygen molecule and the protonated distal histidine at acid pH.     

The interaction of potential-sensitive molecular probes with dimyristoylphosphatidylcholine vesicles investigated by 31P-NMR and electron microscopy

The effect of a number of commonly employed potential-sensitive molecular probes on the 31P-NMR properties of dimyristoylphosphatidylcholine vesicles at two field strengths has been investigated in order to obtain information on the location and effect of these probes on the membrane bilayer. In comparison to the control dye-free vesicle spectrum, the probes diS-C3-(5) and diS-C4-(5), when added to a vesicle suspension, cause a substantial broadening of the 31P resonance with no detectable chemical shift within an uncertainty of +/- 0.05 ppm at 24 MHz. The spin-lattice and spin-spin relaxation times are also reduced when the cyanines are present by well over 20% relative to those of the control vesicle preparation. The addition of anionic probes, including several oxonol derivatives and merocyanine 540, causes no chemical shift, line broadening, or changes in the relaxation times. Possible explanations for the failure of the anionic probes to alter the vesicle 31P-NMR properties include charge repulsion between these dyes and the phosphate group that prevents the probes from penetrating the bilayer to a depth sufficient to alter the local motion of the phosphate moiety. The 31P resonance broadening and reduction in the relaxation times caused by the two cyanines is at least in part due to an increase in vesicle size as judged by electron microscopy measurements, although an inhibition of the local phosphate motion as well cannot be completely eliminated. The cyanine-mediated increase in vesicle size appears to be due to an irreversible vesicle-fusion process possibly initiated by the screening of surface charge by these probes. The implications of these observations in relation to functional energy-transducing preparations is discussed.     

Phosphorus-31 NMR studies of E. coli ribosomes.

Phosphorus-31 nuclear magnetic resonance spectra, relaxation times and nuclear Overhauser (NOE) enhancement have been measured for E. coli ribosomes, subunits and rRNA. NOE and T1 experiments reveal that the phosphorus relaxation in this organelle is largely dipolar in origin. Moreover these results imply the presence of internal motion within the RNA chain with a correlation time of about 3-5 x 10(-9) sec. In all cases the predominant resonance is centered at about -1.5 ppm (relative to 85% H3PO4) as expected for a phosphodiester linkage where there is a large degree of double helix. The linewidth narrows by about a factor of four when the ribosomal proteins are removed indicating a substantial immobilization of the RNA when it is assembled into the ribosome. In addition to the phosphodiester resonance, ribosomes also reveal one or two narrower resonances shifted to low field by 1-4 ppm. Based on the observation that these resonances show a pH dependent chemical shift, we assign them to phosphate monoesters i.e. terminal 3' or 5' phosphate groups. These terminal phosphates are due to short oligomers of RNA derived from the terminus of the chain.     

The Nature and Origin of Chemical Shift for Intracellular Water Nuclei in Artemia Cysts

We investigated the possible existence of chemical shift of water nuclei in Artemia cysts using high resolution nuclear magnetic resonance (NMR) methods. The results conducted at 60, 200, and 500 MHz revealed an unusually large chemical shift for intracellular water protons. After correcting for bulk susceptibility effects, a residual downfield chemical shift of 0.11 ppm was observed in fully hydrated cysts. Similar results have been observed for the deuterium and ¹⁷O nuclei.

We have ruled out unusual intracellular pH, diamagnetic susceptibility of intracellular water, or interaction of water molecules with lipids, glycerol, and/or trehalose as possible origins of the residual chemical shift. We conclude that the residual chemical shift observed for water nuclei (¹H, ²H, and ¹⁷O) is due to significant water-macromolecular interactions.

     

Solid-state nuclear magnetic resonance derived model for dynamics in the polypeptide backbone of the gramicidin A channel.

The dynamics of the backbone of the gramicidin A transmembrane cation channel in dimyristoylphosphatidylcholine bilayers have been investigated using solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopy. With the temperature-dependent fluidity of the bilayer, the rates of motions in the helical gramicidin channel can be modulated. It is shown that in the gel phase, all substantial motions of the channel are slow on the timescale of the n.m.r. experiment (3.5 kHz). The use of oriented samples in which the axis of global channel rotation is aligned parallel to the magnetic field enables separation of global and local dynamics. Spectra obtained from oriented bilayer samples containing single-site 15N-labeled gramicidin at 8 degrees C are analyzed to yield a spatial model for local backbone motion. This model includes the axis of motion, the mean orientation, and the maximum amplitude of displacement for individual peptide planes. Specific sites in the first turn of the amino terminus were investigated, with emphasis on the Ala3 and Leu4 linkages, for which the orientation of the 15N chemical shift tensor with respect to the molecular frame has been determined. The effect of two well-characterized bilayer defect structures, parabolic focal conics and oily streaks, is included in the spectral simulations. It is found that only relatively small amplitude motions are possible at the two sites, with amplitudes of not more than +/- 8 degrees and +/- 15 degrees for the Ala3 and Leu4 sites, respectively. Detailed characterization of the bilayer surface geometry in the oriented samples is presently the major limiting factor in the use of this technique for probing the spatial extent of local motions in integral membrane proteins.     

Influence of dilution on the physical state of model bile systems: NMR and quasi-elastic light-scattering investigations

Multinuclear (1H and 31P) nuclear magnetic resonance (NMR) spectroscopy and quasi-elastic light scattering have been used to characterize molecular aggregates formed in dilute sodium taurocholate--egg lecithin solutions. When mixed micelles (1.25 g/dL) are diluted with 150 mM aqueous sodium chloride, light-scattering measurements suggest a transformation from mixed micelles to unilamellar vesicle species. Decreased 1H NMR line widths for bile salt resonances are consistent with predominance of a monomer form. The concurrent appearance of a second phospholipid choline methyl resonance indicates two types of phospholipid environment in slow chemical exchange: this behavior is consistent with small unilamellar vesicles. The appearance of bilayer vesicles in dilute model bile solutions is confirmed by addition of a lanthanide shift reagent (Pr3+), which splits the 1H or 31P head-group peak into two components with distinct chemical shift sensitivities. These mixed micelle and vesicle aggregates are also distinguished by their susceptibility to the lipolytic enzyme phospholipase A2 from cobra venom.     

Phosphorus-31 nuclear magnetic resonance of aspartate aminotransferase from chicken heart cytosol

31P-nuclear magnetic resonance and absorption spectra of cytosolic chicken aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been recorded in the pH range from 5 to 8.5. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; the chemical shift change was 0.35 ppm. The pK value found by spectrophotometric titration of the enzyme proved to be about 6.0. The monoanion-dianion transition of the 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in the 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein-bound coenzyme is in a dianionic form throughout the investigated pH range; the pH-dependence of the 31P chemical shift may be due to a conformational change at the active site. In the presence of 100 mM succinate, 6 mM aminooxyacetate or 25 mM cycloserine, the 31P chemical shift is insensitive to pH variations.