Hyperfine shift NMR studies of hydrated phospholipid inverted micelles

The ³¹P nuclear magnetic resonance (NMR) spectra of benzene solutions of hydrated dipalmitoyl lecithin (DPL) inverted micelles, with and without incorporated paramagnetic lanthanide ions, have been recorded. Individual resonances for micelles containing none, one, and two ions can be resolved and observed in the presence of one another. The relative intensities of these peaks yield some information on the state of aggregation of lipid inverted micelles prepared by ultrasonic irradiation. The relative intensities and chemical shifts of resonances of unsonicated mixtures of preformed micelles containing different numbers of ions per micelle indicate that some kind of equilibration occurs. The data are consistent with a selective fusion of multi-ion micelles with ion-free micelles. The NMR spectra place constraints on the lifetimes of metal ions and lipid and water molecules within a micelle before transfer to another.     

Conformation of membrane-associated proapoptotic tBid

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.     

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.     

Delta-opiate DPDPE in magnetically oriented phospholipid micelles: binding and arrangement of aromatic pharmacophores

D-Penicillamine(2,5)-enkephalin (DPDPE) is a potent opioid peptide that exhibits a high selectivity for the delta-opiate receptors. This zwitterionic peptide has been shown, by pulsed-field gradient 1H NMR diffusion studies, to have significant affinity for a zwitterionic phospholipid bilayer. The bilayer lipid is in the form of micelles composed of dihexanoylphosphatidylcholine (DHPC) and dimyristoylphosphatidylcholine (DMPC) mixtures, where the DMPC forms the bilayer structure. At high lipid concentration (25% w/w) these micelles orient in the magnetic field of an NMR spectrometer. The resulting 1H-13C dipolar couplings and chemical shift changes in the natural abundance 13C resonances for the Tyr and Phe aromatic rings were used to characterize the orientations in the bilayer micelles of these two key pharmacophores.     

Interaction of an amine oxide detergent with lecithin vesicles as studied by nuclear magnetic resonance

The interaction of an amine oxide detergent with single bilayer lecithin vesicles was investigated with proton and phosphorus magnetic resonance. The addition of the detergent micelles to vesicles suspensions leads to rapid detergent incorporation into the vesicle bilayer, resulting in a heterogenous vesicle population. Initially, some vesicles take up the equivalent of one detergent micelle, whereas others contain no detergent. Subsequently, the detergent is distributed between the vesicles by vesicle-vesicle collisions. This can be followed by the change in the Pr3+-shifted spectral positions of the detergent and lecithin head groups with time. From the intensity of the head-group signals, it can be concluded that after about 20 h the detergent is almost equally distributed between the outer and inner vesicle membrane monolayers. Vesicles obtained by cosonication of the detergent and lecithin take up metal ions. This ion permeability depends on the vesicle concentration and can be attributed to vesicle-vesicle or vesicle-mixed micelle collisions. Egg lecithin vesicles are stable against the detergent up to molar ratios of detergent to lecithin of 0.2--0.3. At larger ratios mixed micells and multibilayers are formed. Measurements of proton spin-lattice relaxation times confirmed that the internal architecture of the vesicle bilayer is almost unaffected by the incorporated detergent.     

Transmembrane helix-helix interactions: comparative simulations of the glycophorin a dimer

The glycophorin helix dimer is a paradigm for the exploration of helix-helix interactions in integral membrane proteins. Two NMR structures of the dimer are known, one in a detergent micelle and one in a lipid bilayer. Multiple (4 x 50 ns) molecular dynamics simulations starting from each of the two NMR structures, with each structure in either a dodecyl phosphocholine (DPC) micelle or a dimyristoyl phosphatidylcholine (DMPC) bilayer, have been used to explore the conformational dynamics of the helix dimer. Analysis of the helix-helix interaction, mediated by the GxxxG sequence motif, suggests convergence of the simulations to a common model. This is closer to the NMR structure determined in a bilayer than to micelle structure. The stable dimer interface in the final simulation model is characterized by (i) Gly/Gly packing and (ii) Thr/Thr interhelix H-bonds. These results demonstrate the ability of extended molecular dynamics simulations in a lipid bilayer environment to refine membrane protein structures or models derived from experimental data obtained in protein/detergent micelles.     

Helical structure of dermaseptin B2 in a membrane-mimetic environment

Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3-18, are stabilized by addition of TFE. SDS titration showed that dermaseptin B2 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11-31 connected to a more flexible helical segment spanning residues 1-8 by a flexible hinge region around Val9 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1-11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure-activity studies on dermaseptin B2 analogues showed that the N-terminal 1-11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10-33 region is also important for full antibiotic activity.     

Nuclear magnetic resonance studies of cation transport across vesicle bilayer membranes.

We analyze an increasingly popular NMR method analogous to the black lipid membrane (BLM) isotopic tracer experiment for the study of mediated cation transport but involving the preparation of vesicles with an environment asymmetric in that paramagnetic metal ions are present only outside the vesicles. This asymmetry is manifest in the NMR spectrum as two distinct resonances for magnetic nuclei in outside and inside lipid headgroups. As mediated transport begins and for the paramagnetic metal ions enter the vesicles, the inner headgroup resonance line shifts and changes shape with a time course containing much information on the actual ion transport mechanism. Processes by which the ions enter the vesicles one or a few at a time (such as via a diffusive carrier) are easily distinguishable from those by which the ions enter in large bursts (such as by pore activation). The limiting case where intervesicular mediator exchange is slow relative to cation transport (the situation for integral membrane proteins) is treated analytically. Computer simulated curves indicate conditions necessary for certain changes in the line shape which are analogous to the "current jumps" observed in BLM conductance studies. The theory derived allows estimates of the average number of ions entering the first few bursts, how often the bursts occur, and how they depend on the concentration of the mediating species in the vesicular membrane. Preliminary experimental spectra illustrating some of the various possible line shape behaviors are presented.     

NMR studies of the influence of dodecyl sulfate on the amide hydrogen exchange kinetics of a micelle-solubilized hydrophobic tripeptide

Backbone amide hydrogen exchange measurements are an important source of information about the internal dynamics of proteins. Before such measurements can be interpreted unambiguously, contributions to hydrogen exchange rates from the chemical and physical environment of the amides must be taken into account. Membrane proteins are often solubilized in detergents, yet there have not been any systematic investigations of the possible effects detergents may have on the amide hydrogen exchange rates of proteins. To address this question, we have measured individual backbone and carboxyl-terminal amide exchange rates for the amphipathic tripeptide Leu-Val-Ile-amide dissolved in water and dodecyl sulfate micelles. 1H NMR spectroscopy was used to measure exchange using the direct exchange-out into D2O technique at 5 degrees C and using an indirect steady-state saturation-transfer technique at 25 degrees C. The broadening effect of micelle-incorporated spin-labeled fatty acid (12-doxylstearate) on the 1H NMR spectra of both the detergent and the peptide resonances was used to demonstrate that the tripeptide is intimately associated with the micelle. The resonance from formate ion, which is excluded from the micelle, was unperturbed by the spin label. The detergent did not retard the exchange rates of either the primary (terminal) or secondary (backbone) amides of the tripeptide. This suggests that the micelle/peptide interaction does not restrict access of charged catalysts and water to these amides and shows that the peptide amides are not hydrogen bonded. However, the pH for the exchange minima of these amides in detergent was increased between 1.2 and 1.7 units compared to exchange in water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deuterium nuclear magnetic resonance studies of bile salt/phosphatidylcholine mixed micelles

Mixed micelles of deoxycholate (DOC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) have been prepared in which the POPC was specifically deuterated in the 2-, 6-, 10-, or 16-position of the palmitoyl chain or in the N-methyl position of the choline head group. The deuterium nuclear magnetic resonance (2H NMR) spectrum of each of these specifically deuterated mixed micelles consists of a singlet whose line width depends upon the position of deuteration. Spin-spin relaxation times indicate a gradient of mobility along the POPC palmitoyl chain in the mixed micelle, with a large increase in mobility on going from the 10- to the 16-position. Spin-lattice relaxation times (T1's) demonstrate a similar gradient of mobility. Both trends in NMR relaxation behavior are consistent with a bilayer arrangement for the solubilized POPC. 2H T1 times for DOC/POPC micelles are significantly shorter than those measured in other bilayer systems, indicating unusually tight phospholipid acyl chain packing in the mixed micelle.     

1H, 113Cd, and 31P NMR of osteocalcin (bovine gamma-carboxyglutamic acid containing protein)

The 1H (500-MHz), 113Cd (44-MHz), and 31P (81-MHz) NMR spectra of the bovine gamma-carboxyglutamate- (Gla-) containing protein osteocalcin and its Ca(II) and Cd(II) complexes in solution have been obtained. The 1H NMR spectrum of the native protein shows narrow resonances and a highly resolved multiplet structure suggesting rotational freedom of the side chains. In comparison to the simulated 1H NMR spectrum of a random polypeptide chain of the same amino acid composition, there is moderate chemical shift dispersion, indicating some conformational restraints to be present. Ca(II) binding broadens all 1H resonances, so severely at four Ca(II) ions per molecule that few structural conclusions can be made. Cd(II) substituted for Ca(II) has the same effect, and 113Cd NMR shows the Cd(II) to be in intermediate chemical exchange on the chemical shift time scale. Estimates of the chemical exchange rates required for 1H and 113Cd line broadening suggest a range of Kd values for the metal ion complexes from 10(-6) M to as high as 10(-3) M depending on the number of metal ions bound. Alternatively, 1H line broadening could be explained by relatively slow conformational fluxes in the protein induced by labile metal ion binding to one or more sites. Cd(II) when used to form a cadmium-phosphate mineral analogous to hydroxylapatite results in a crystal lattice that removes osteocalcin from solution just as effectively as hydroxylapatite. 113Cd(II) exchange at the binding sites of osteocalcin in solution is slowed dramatically by the addition of HPO4(2-). 31P NMR shows the interaction of phosphate with the protein to require the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal binding sites of a gamma-carboxyglutamic acid-rich fragment of bovine prothrombin.

The metal binding sites of a gamma-carboxyglutamic acid-rich fragment derived from bovine prothrombin were examined using paramagnetic lanthanide ions to evaluate the role of gamma-carboxyglutamic acid resideus in metal binding. A gamma-carboxyglutamic acid-rich peptide, fragment 12-44, was isolated from a tryptic digest of prothrombin. Using 153Gd(III), fragment 12-44 was found to contain one high affinity metal binding site (KD = 0.55 microM) and four to six lower affinity metal binding sites (KD approximately 4 to 8 microM). The S-carboxymethyl derivative of fragment 12-44, in which the disulfide bond in fragment 12-44 was reduced and alkylated, contained no high affinity metal binding site and four or five lower affinity sites (KD = 8 microM). The effects of paramagnetic lanthanide ions on fragment 12-44 and its S-carboxymethyl derivative were studied by natural abundance 13C NMR spectroscopy. The 13C NMR spectrum of fragment 12-44 was recorded at 67.88 MHz and the resonances were assigned by comparison to the chemical shift of carbon resonances of amino acids and peptides previously studied. The proximity between bound metal ions and carbon atoms in fragment 12-44 was estimated using Gd(III), based upon the strategy that the magnitude of the change in the transverse relaxation rate of resonances of carbon nuclei induced by bound metal ions is related in part to the interatomic distances between bound metal and carbon nuclei. Titration of fragment 12-44 with Gd(III) resulted in the selective broadening of the gamma-carboxyl carbon, C gamma, C beta, and C alpha resonances of gamma-carboxyglutamic acid, and the C epsilon of the arginines. S-Carboxymethyl fragment 12-44, which lacked the high affinity metal binding site, showed markedly decreased perturbation of the C epsilon of the arginine residues upon titration with Gd(III). These studies indicate that gamma-carboxyglutamic acid residues in prothrombin fragment 12-44 participate in metal liganding. A high affinity metal binding site in fragment 12-44 is in close proximity of Arg 16 and Arg 25 and is stabilized by the disulfide bond. On the basis of these data, a model of the metal binding sites is proposed in which the high affinity site is composed of two gamma-carboxyglutamic acid residues which participate in intramolecular metal-dependent bridging of two regions of the polypeptide chain. The lower affinity metal binding sites, formed by single or paired adjacent gamma-carboxyglutamic acid residues, then may participate in intermolecular metal-dependent protein . protein or protein . membrane complex formation.     

Binding and folding of melittin in the presence of ganglioside GM1 micelles

In this work we examine the binding and folding of the membrane-active peptide, melittin in the presence of ganglioside GM1 micelle. The membrane bilayer is capable of inducing folding to small proteins and peptides upon binding. Using two-dimensional NMR techniques we have shown that at low concentration, GM1 micelle is able to induce an extended helical conformation to MLT. The pulsed-field gradient diffusion NMR study indicates that the peptide partition into GM1 micelle along with about 32% binding. While looking for the binding between MLT and GM1 using saturation transfer difference NMR spectroscopy, Val5, Leu9, Thr11, Ile17, Ser18, and Trp19 have been identified as the residues that are in close proximity to GM1 micelles.     

Lipid exchange between mixed micelles of phospholipid and triton X-100

If phospholipase catalyzed hydrolysis of phospholipid dissolved in a detergent mixed micelle is limited to the phospholipid carried by a single micelle, then hydrolysis ceases upon exhaustion of that pool. However, if the rate of phospholipid exchange between micelles exceeds the catalytic rate then all of the phospholipid is available for hydrolysis. To determine phospholipid availability we studied the exchange of 1,2-dioleoyl-sn-glycero-3-phosphocholine between mixed micelles of phospholipid and non-ionic Triton detergents by both stopped-flow fluorescence-recovery and nuclear magnetic resonance-relaxation techniques. Stopped-flow analysis was performed by combining mixed micelles of Triton and phospholipid with mixed micelles that contained the fluorescent phospholipid 1-palmitoyl-2-(12-[{7-nitro-2-1, 3-benzoxadiazo-4-yl}amino]dodecanoyl)-sn-glycero-3-phosphocholine (P-2-NBD-PC). The concentration dependence of fluorescence recovery suggested a second-order exchange mechanism that was saturable. The true second-order rate constant depends on the specific mechanism for exchange, which was not determined in this study, but the rate constant will be on the order of 106 to 107 M-1s-1. Incorporation of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine into micelles increased the rate of proton relaxation and gave a limiting relaxation time of 1.3 ms. The results demonstrate that phospholipid exchange was rapid and that the phospholipid content of a single micelle did not limit the rate of phospholipid hydrolysis by phospholipases.     

Conformation and environment of channel-forming peptides: a simulation study

Ion channel-forming peptides enable us to study the conformational dynamics of a transmembrane helix as a function of sequence and environment. Molecular dynamics simulations are used to study the conformation and dynamics of three 22-residue peptides derived from the second transmembrane domain of the glycine receptor (NK4-M2GlyR-p22). Simulations are performed on the peptide in four different environments: trifluoroethanol/water; SDS micelles; DPC micelles; and a DMPC bilayer. A hierarchy of alpha-helix stabilization between the different environments is observed such that TFE/water < micelles < bilayers. Local clustering of trifluoroethanol molecules around the peptide appears to help stabilize an alpha-helical conformation. Single (S22W) and double (S22W,T19R) substitutions at the C-terminus of NK4-M2GlyR-p22 help to stabilize a helical conformation in the micelle and bilayer environments. This correlates with the ability of the W22 and R19 side chains to form H-bonds with the headgroups of lipid or detergent molecules. This study provides a first atomic resolution comparison of the structure and dynamics of NK4-M2GlyR-p22 peptides in membrane and membrane-mimetic environments, paralleling NMR and functional studies of these peptides.     

Enhancement of RNA self-cleavage by micellar catalysis.

It has been reported recently that naturally occurring catalytic RNAs like hammerhead and hairpin ribozyme do not require metal ions for efficient catalysis. It seems that the folded tertiary structure of the RNA contributes more to the catalytic function than was initially recognized. We found that a highly specific self-cleavage reaction can occur within a small bulge loop of four nucleotides in a mini-substrate derived from Arabidopsis thaliana intron-containing pre-tRNA(Tyr) in the absence of metal ions. NH(4)(+) cations and non-ionic or zwitter-ionic detergents at or above their critical micelle concentration are sufficient to catalyze this reaction. The dependence on micelles for the reaction leads to the assumption that physical properties, i.e. the hydrophobic interior of a micelle, are essential for this self-cleavage reaction. We suggest that NH(4)(+)-ions play a crucial role for the entry of the negatively charged RNA into the hydrophobic interior of a detergent micelle. A change of the pattern of hydration or hydrogen bonds caused by the hydrophobic surrounding enhances the reaction by a factor of 100. These findings suggest that highly structured RNAs may shift pK(a) values towards neutrality via the local environment and thereby enhance their ability to perform general acid-base catalysis without the participation of metal ions.     

Mixed micelles and other structures in the solubilization of bilayer lipid membranes by surfactants

The solubilization of lipid bilayers by surfactants is accompanied by morphological changes of the bilayer and the emergence of mixed micelles. From a phase equilibrium perspective, the lipid/surfactant/water system is in a two-phase area during the solubilization: a phase containing mixed micelles is in equilibrium with bilayer structures of the lamellar phase. In some cases three phases are present, the single micelle phase replaced by a concentrated and a dilute solution phase. In the case of non-ionic surfactants, the lipid bilayers reach saturation when mixed micelles, often flexible rod-like or thread-like, start to form in the aqueous solution, at a constant chemical potential of the surfactant. The composition of the bilayers also remains fixed during the dissolution. The phase behavior encountered with many charged surfactants is different. The lamellar phase becomes destabilized at a certain content of surfactant in the membrane, and then disintegrates, forming mixed micelles, or a hexagonal phase, or an intermediate phase. Defective bilayer intermediates, such as perforated vesicles, have been found in several systems, mainly with charged surfactants. The perforated membranes, in some systems, go over into thread-like micelles via lace-like structures, often without a clear two-phase region. Intermediates in the form of disks, either micelles or bilayer fragments, have been observed in several cases. Most noteworthy are the planar and circular disks found in systems containing a large fraction of cholesterol in the bilayer. Bile salts are a special class of surfactants that seem to break down the bilayer at low additions. Originally, disk-like mixed micelles were conjectured, with polar membrane lipids building the disk, and the bile salts covering the hydrophobic rim. Later work has shown that flexible cylinders are the dominant intermediates also in these systems, even if the disk-like structures have been re-established as transients in the transformation from mixed micelles to vesicles.     

NMR evidence for progressive stabilization of native-like structure upon aggregation of acid-denatured LysN

The acid-denatured form of the protein LysN aggregates reversibly at pH 2.0. The strength of self-association increases with increasing Cl(-) anion concentration. At low concentrations of protein or Cl(-) anion, resonances of denatured LysN are in slow exchange with a minor form of the protein, which shows native-like NMR chemical shifts. The minor native-like resonances increase in intensity with increasing protein concentration, demonstrating that a native-like monomer fold is stabilized on aggregation of the acid-denatured protein. At high concentrations of protein or Cl(-) anion, interconversion between the major and minor resonances appears to shift from slow to intermediate exchange on the NMR timescale. NMR line-broadening is more pronounced for the major resonances of the denatured protein, which show sigmoidal decay curves with increasing Cl(-) concentration. The mid-points of the decay curves for residues in different parts of the molecule are non-coincident. We propose that differences in the NMR line-broadening transitions of individual residues reflect a stepwise stabilization of native-like structure on aggregation, starting with the segments of the protein that form the initial association interface. The resonances of the denatured protein with the greatest sensitivity to self-association correspond roughly to those that are most perturbed in the native protein on binding of the natural substrate tRNA(Lys). This suggests that the hydrophobic surfaces that promote intermolecular misfolding of acid-denatured LysN, may resemble those used for substrate binding by the native protein.     

Oncomodulin. 1H NMR and optical stopped-flow spectroscopic studies of its solution conformation and metal-binding properties

As deduced from its 1H NMR spectrum, oncomodulin's solution conformation is very similar to the tertiary structure of other single domain 2-site calcium-binding proteins of the troponin C class. Despite its extensive amino acid sequence homology with parvalbumins, however, oncomodulin differs significantly from these proteins in its Ca(II)----Ln(III) exchange characteristics. Although the relative affinity of Lu(III) for the EF site of Ca2-oncomodulin was normal, beta Lu:EF/beta Ca:EF being 175 +/- 15, displacement of Ca(II) from the CD site was not favored, beta Lu:CD/beta Ca:CD being 1.2 +/- 0.1. Lineshape analyses of several 1H NMR resonances generated by the Lu(III) titration of Ca2-oncomodulin indicated that Ca(II)----Ln(III) exchange at the CD site was 15-20 s-1, approximately 100 times faster than exchange at the CD site of parvalbumins. Analyses of the distribution of metal-bound oncomodulin species showed that Ca(II)----Lu(III) exchange was cooperative, the coefficient of cooperativity being estimated as 5 +/- 1. The kinetics of the release of Yb(III) from oncomodulin as measured by optical stopped-flow techniques corroborated the observed cooperativity in metal binding; the off-rate constant of Yb(III) from the EF site of Yb2-oncomodulin was 0.0036 s-1, approximately 19 times slower than the release of Yb(III) from the EF site of Ca1Yb1-oncomodulin. We attribute part of the reduced preference of small Ln(III)s for the CD site of oncomodulin to a combination of this site's inherent incompressibility (Williams, T.C., Corson, D.C. & Sykes, B.D. (1984) J. Am. Chem. Soc. 106, 5698-5702) and the Glu----Asp substitution at sequence position 59, the residue which chelates metal at the -X coordination position. Like the CD site in oncomodulin, site III in troponin C has not only a lower affinity for calcium relative to the CD site of parvalbumins but also aspartic acid at its -X position; a water molecule bridges the gap between bound metal and the carboxyl group of the relatively short side chain of Asp-114 (Herzberg, O. & James, M. N. G. (1985) Biochemistry 24, 5298-5302). Hence, we suggest that Asp-59 in oncomodulin binds metal only indirectly through an intervening water molecule, a proposal which is consistent with the CD site's reduced affinity for ions the size of Ca(II) or smaller.     

Polar group conformation of 1,2-di-O-alkylglycerophosphocholines in the absence and presence of ions

The conformation of the glycerophosphocholine (GPC) group of various 1,2-di-O-alkyl and 1,2-diacylglycerophosphocholines forming small micelles or single-bilayer vesicles in H₂O has been studed by NMR in the absence and presence of lanthanide ions. In the absence of lanthanides the motionally averaged polar group conformation of 1,2-di-O-alkylglycerophosphocholine (dialkyl-GPC) is similar to that of the diacyl compound. The replacement of the ester linkages in diacyl phosphatidylcholine by ether bonds has therefore no significant effect on the conformation and segmental motion of the glycerophosphocholine group. This conformation is found to be independent of the state of aggregation, i.e., the main features are the same below and above the critical micellar concentration (CMC). The determining factor must therefore be the intramolecular energetics. Within the experimental accuracy the conformation of dialkyl-GPC in the presence of lanthanide ions is also the same as that of the corresponding diacyl compound. Furthermore, in the presence of lanthanides the polar group conformation of dialkyl-GPC is the same within experimental accuracy in small micelles and single bilayer vesicles. The conformational change induced by lanthanides leads to a reorientation of the OPN dipole. In the presence of lanthanides the OPN dipole increases its angle of tilt with respect to the bilayer plane from about 0° (coplanar orientation) to an average inclinication of about 45°. This gives rise to a more extended disposition of the polar group with respect to the bilayer normal.