Secondary structure of human apolipoprotein A-I(1-186) in lipid-mimetic solution.

The solution structure of an apoA-I deletion mutant, apoA-I(1-186) was determined by the chemical shift index (CSI) method and the torsion angle likelihood obtained from shift and sequence similarity (TALOS) method, using heteronuclear multidimensional NMR spectra of [u-(13)C, u-(15)N, u-50% (2)H]apoA-I(1-186) in the presence of sodium dodecyl sulfate (SDS). The backbone resonances were assigned from a combination of triple-resonance data (HNCO, HNCA, HN(CO)CA, HN(CA)CO and HN(COCA)HA), and intraresidue and sequential NOEs (three-dimensional (3D) and four-dimensional (4D) 13C- and 15N-edited NOESY). Analysis of the NOEs, H(alpha), C(alpha) and C' chemical shifts shows that apoA-I(1-186) in lipid-mimetic solution is composed of alpha-helices (which include the residues 8-32, 45-64, 67-77, 83-87, 90-97, 100-140, 146-162, and 166-181), interrupted by short irregular segments. There is one relatively long, irregular and mostly flexible region (residues 33-44), that separates the N-terminal domain (residues 1-32) from the main body of protein. In addition, we report, for the first time, the structure of the N-terminal domain of apoA-I in a lipid-mimetic environment. Its structure (alpha-helix 8-32 and flexible linker 33-44) would suggest that this domain is structurally, and possibly functionally, separated from the other part of the molecule.

Structural studies of a baboon (Papio sp.) plasma protein inhibitor of cholesteryl ester transferase.

A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in baboons (Papio sp.). The cholesteryl ester transfer inhibitor protein (CETIP) corresponds to the N-terminus of baboon apoC-I. Relative to CETIP, baboon apoC-I is a weak inhibitor of baboon cholesteryl ester transferase (CET). To study the structural features responsible for CET inhibition, CETIP was synthesized by solid-phase methods. Using sodium dodecyl sulfate (SDS) to model the lipoprotein environment, the solution structure of CETIP was probed by optical and 1H NMR spectroscopy. Circular dichroism data show that the protein lacks a well-defined structure in water but, upon the addition of SDS, becomes helical (56%). A small blue shift of 8 nm was observed in the intrinsic tryptophan fluorescence of CETIP in the presence of saturating amounts of SDS, suggesting that tryptophan-23 is not buried deeply in the lipid environment. The helical nature of CETIP in the presence of SDS was confirmed by upfield 1Halpha secondary shifts and an average solution structure determined by distance geometry/simulated annealing calculations using 476 NOE-based distance restraints. The backbone (N-Calpha-C=O) root-mean-square deviation of an ensemble of 17 out of 25 calculated structures superimposed on the average structure was 1.06+0.30 A using residues V4-P35 and 0.51+/-0.17 A using residues A7-S32. Although the side-chain orientations fit the basic description of a class A amphipathic helix, both intramolecular salt bridge formation and "snorkeling" of basic side chains toward the polar face play minor, if any, roles in stabilizing the lipid-bound amphipathic structure. Conformational features of the calculated structures for CETIP are discussed relative to models of CETIP inhibition of cholesteryl ester transferase.

Structure of a biologically active fragment of human serum apolipoprotein C-II in the presence of sodium dodecyl sulfate and dodecylphosphocholine.

We have studied the three-dimensional structure of a biologically active peptide of apolipoprotein C-II (apoC-II) in the presence of lipid mimetics by CD and NMR spectroscopy. This peptide, corresponding to residues 44-79 of apoC-II, has been shown to reverse the symptoms of genetic apoC-II deficiency in a human subject. A comparison of alpha-proton secondary shifts and CD spectroscopic data indicates that the structure of apoC-II(44-79) is similar in the presence of dodecylphosphocholine and sodium dodecyl sulfate. The three-dimensional structure of apoC-II(44-79) in the presence of sodium dodecyl sulfate, determined by relaxation matrix calculations, contains two amphipathic helical domains formed by residues 50-58 and 67-75, separated by a non-helical linker centered at Tyr63. The C-terminal helix is terminated by a loop formed by residues 76-79. The C-terminal helix is better defined and has a larger hydrophobic face than the N-terminal helix, which leads us to propose that the C-terminal helix together with the non-helical Ile66 constitute the primary lipid binding domain of apoC-II(44-79). Based on our structure we suggest a new mechanism of lipoprotein lipase activation in which both helices of apoC-II(44-79) remain lipid bound, while the seven-residue interhelical linker extends away from the lipid surface in order to project Tyr63 into the apoC-II binding site of lipoprotein lipase.

Conformation of human apolipoprotein C-I in a lipid-mimetic environment determined by CD and NMR spectroscopy.

The high-resolution conformation of human apoC-I in complexes with sodium dodecyl sulfate (SDS) is presented. As estimated from CD data, apoC-I adopts 54% helical secondary structure when bound to SDS, which is similar to the helical content previously found with phospholipids. The NMR-derived conformation of apoC-I is composed of two amphipathic helices, residues 7-29 and 38-52, separated by a flexible linker. The N-terminal helix contains a mobile hinge involving residues 12-15. The hydrophobic side chains cluster on the nonpolar face of both helices, thus forming two discrete lipid-binding sites in the N-terminal helix and one in the C-terminal helix. As suggested by amide proton resonance line widths and deuterium exchange rates, the N-terminal helix is more flexible and may bind less tightly to the detergent than the C-terminal helix. The different mobility of both helices appears to be related to side-chain composition, rather than length of the amphipathic helix, and may play a role in the function of apoC-I as an activator of lecithin:cholesterol acyltransferase (LCAT). A model is suggested in which the C-terminal helix serves as a lipid anchor while the N-terminal helix may hinge off the lipid surface to make specific contacts with LCAT.

The use of sodium dodecyl sulfate to model the apolipoprotein environment. Evidence for peptide-SDS complexes using pulsed-field-gradient NMR spectroscopy.

Pulsed-field-gradient NMR spectroscopy was used to measure translational diffusion coefficients (Ds) for a peptide corresponding to a proposed lipid-binding domain of human apolipoprotein C-I, residues 7-24 (apoC-I(7-24)). Diffusion coefficients for apoC-I(7-24) were determined directly by following the decay of the resonance intensity of selected peptide protons at various concentrations of sodium dodecyl sulfate (SDS), a detergent increasingly being used to model the apolipoprotein environment. Previously, diffusion coefficients of peptides in the presence of SDS have been determined indirectly by monitoring the SDS diffusion coefficient. The direct measurement of the diffusion coefficient of the peptide enables one to distinguish whether SDS simply coats the peptide's surface to produce a uniformly charged 'rod' or if the peptide associates with a micelle. Using the direct method, at SDS concentrations above 5 mM (which is below the SDS critical micelle concentration (8.1 mM)), apoC-I(7-24) exhibited diffusion coefficients consistent with the formation of a large-molecular-weight complex. Based on the ratio of the diffusion coefficients for free- and SDS-associated peptide, the molecular weight of the peptide-SDS complex was much larger than a factor of 1. 4, the increase in molecular weight of the free peptide predicted if apoC-I(7-24) was uniformly surface coated with SDS.

Sequence-specific 1H NMR resonance assignments and secondary structure of human apolipoprotein C-I in the presence of sodium dodecyl sulfate.

Apolipoprotein (apo) C-I is a 57-residue exchangeable plasma protein distributed mainly in high and very low density lipoprotein. In this report we present the nuclear magnetic resonance spectra of native apoC-I and synthetic apoC-I, containing selected 15N-labelled amino acids, in the presence of sodium dodecyl sulfate. The proton resonances of apoC-I are assigned and the secondary structure is estimated from the difference of measured alpha-proton chemical shifts to random coil values and the observed NOE interactions. According to these data apoC-I forms two helices, Val-4-Lys-30 and Leu-34-Lys-52, linked by an unstructured region Gln-31-Glu-33. The N-terminal segments of each helix, Val-4-Gly-15 and Leu-34-Met-38, appear to be more flexible than the helical core regions Asn-16-Lys-30 and Arg-39-Lys-52.

Infrared spectroscopy of human apolipoprotein fragments in SDS/D2O: relative lipid-binding affinities and a novel amide I assignment.

Infrared absorption spectra are reported for six apolipoprotein fragments in SDS/D2O. Five of the peptides correspond to proposed lipid-binding domains of human apolipoproteins [apoC-I(7-24), apoC-I(35-53), apoA-II(18-30)+, apoA-I(166-185), apoE(267-289)], and the sixth is the de novo lipid associating peptide LAP-20. The amide I infrared absorption patterns are generally consistent with predominantly helical structures (as determined previously by NMR spectroscopy and distance geometry calculations) and further suggest that apoA-I(166-185) and apoE(267-289) are bound to SDS relatively weakly in comparison to the other four peptides. The latter conclusion is also supported by the temperature dependence of the infrared spectra, as increasing temperature promotes a distinct increase in random coil structure only for apoA-I(166-185) and apoE(267-289). In addition to features readily ascribed to helices, the infrared spectra of all the peptides show absorptions in the spectral region 1630-1635 cm-1 that is usually associated with beta-structure, a motif that is clearly absent from the NMR-derived structures. Parallel difficulties also arose in the analyses of the circular dichroism spectra. We suggest that both the low-frequency infrared absorptions and the ambiguities in interpreting the CD spectra may be due to unusual structures at the peptide C-termini, involving C=O groups that form hydrogen bonds simultaneously either with two solvent molecules or with donors from the backbone (NH) and the solvent (OH). Analogous absorptions may be a general feature of solvent-exposed helices, which suggests a need for caution in assigning amide I bands below 1640 cm-1.

The helix-hinge-helix structural motif in human apolipoprotein A-I determined by NMR spectroscopy.

The conformation of a synthetic peptide of 46 residues from apoA-I was investigated by fluorescence, CD, and 2D NMR spectroscopies in lipid-mimetic environments. ApoA-I(142-187) is mainly unstructured in water but helical in SDS or dodecylphosphocholine (DPC), although the peptide only associates with DPC at approximately the critical micellar concentration. Solution structures of apoA-I(142-187) were determined by distance geometry calculations based on 450 (in DPC-d38) or 397 (in SDS-d25) NOE-derived distance restraints, respectively. Backbone RMSDs for superimposing the two helical regions 146-162 and 168-182 are 0.98 +/- 0.22 (2.38 +/- 0.20) and 1.99 +/- 0.42 (2.02 +/- 0.21) A in DPC (SDS), respectively. No interhelical NOE was found, suggesting that helix-helix interactions between the two helical domains in apoA-I(142-187) are unlikely. Similar average, curved helix-hinge-helix structures were found in both SDS and DPC micelles with the hydrophobic residues occupying the concave face, indicating that hydrophobic interactions dominate. Intermolecular NOESY experiments, performed in the presence of 50% protonated SDS, confirm that the two amphipathic helices and Y166 in the hinge all interact with the micelle. The involvement of Y166 in lipid binding is supported by fluorescence spectroscopy as well. On the basis of all the data above, we propose a model for the peptide-lipid complexes wherein the curved amphipathic helix-hinge-helix structural motif straddles the micelle. The peptide-aided signal assignment achieved for apoA-I(122-187) (66mer) and apoA-I suggests that such a structural motif is retained in the longer peptide and most likely in the intact protein.

Conformational studies of the N-terminal lipid-associating domain of human apolipoprotein C-I by CD and 1H NMR spectroscopy.

A peptide comprising the N-terminal 38 residues of human apolipoprotein C-I (apoC-I(1-38)) was synthesized using solid-phase methods and its solution conformation studied by CD and 1H NMR spectroscopy. The CD data indicate that apoC-I(1-38) has a similar helical content (55%) in the presence of saturating amounts of SDS or egg yolk lysophosphatidylcholine. A structural ensemble of SDS-bound apoC-I(1-38) was calculated from 464 NOE-based distance restraints using distance geometry methods. ApoC-I(1-38) adopts a helical structure between residues V4 and K30 and an extended C-terminus from Q31 when associated with SDS. The region K12-G15 undergoes slow conformational exchange as indicated by above-average amide resonance linewidths, large temperature coefficients, and fast exchange (< 2 h) of backbone amide protons with deuterium. The mobility of K12-G15 is reflected in the poorly defined dihedral angles of K12 and E13 in the calculated ensemble of structures. The average structure of apoC-I(1-38) is curved toward its hydrophobic face with bends of 125 degrees, centered at K12/E13, and 150 degrees, centered at K21. This curvature appears to be driven by the interaction of two hydrophobic clusters, one formed by residues L8, L11, F14, and L18, and the other by L25, I26, and I29, with the amphiphile SDS. Based on our present structural definition of apoC-I(1-38) and the previously obtained structure of the fragment apoC-I(35-53), we propose the secondary structure of intact apolipoprotein C-I.

Conformations of human apolipoprotein E(263-286) and E(267-289) in aqueous solutions of sodium dodecyl sulfate by CD and 1H NMR.

Structures of apoE(263-286) and apoE(267-289) have been determined in aqueous solution containing 90-fold molar excess of perdeuterated sodium dodecyl sulfate by CD and 1H NMR. Conformations were calculated by distance geometry based on 370 and 276 NOE distance restraints, respectively. RMSD for superimposing the region 265-284 from an ensemble of 41 structures for apoE(263-286) is 0.64 +/- 0.17 A for backbone atoms (N, C alpha, C = O) and 1.51 +/- 0.13 A for all atoms. The backbone RMSD for an ensemble of 37 structures for apoE(267-289) is 0.74 +/- 0.21 A for the region 268-275 and 0.34 +/- 0.10 A for the region 276-286. A two-domain structure was found for apoE(267-289) with the C-terminal half adopting a very well defined helix and the N-terminal segment 268-275 a less well defined helix, suggesting that the N-terminus may weakly bind to SDS. For apoE(263-286), an amphipathic helix-bend-helix structural motif was found with all hydrophobic side chains on the concave face. The existence of a bend around residues Q273 to G278 is consistent with their temperature coefficients of amide protons as well as secondary shifts of alpha-protons. Comparison of the structures of the two peptides revealed that the enhanced binding of apoE(263-286) to lipid could be attributed to the formation of a hydrophobic cluster consisting of residues W264, F265, L268, and V269. Aromatic side chains are proposed to be especially important in anchoring apolipoprotein fragments to micelles.

Conformational studies of an amphipathic peptide corresponding to human apolipoprotein A-II residues 18-30 with a C-terminal lipid binding motif EWLNS.

A peptide was designed and synthesized to enhance the lipid binding properties of a 13-residue fragment of apolipoprotein A-II. The peptide, VTDYGKDLMEKVKEWLNS [apoA-II(18-30)+], contains a five-residue amphipathic motif, EWLNS, at the C-terminus of apolipoprotein A-II residues 18-30. The lipid binding properties of apoA-II(18-30)+ were assessed using optical spectroscopy in the presence of sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), tetradecyltrimethyl ammonium chloride (TMA) and dimyristoylphosphatidylcholine (DMPC). The fluorescence emission spectra and the circular dichroism data suggested that apoA-II(18-30)+ interacted most strongly with SDS and most weakly with DMPC. An ensemble of structures for apoA-II(18-30)+ in aqueous solution containing SDS was calculated using distance geometry/simulated annealing methods from 308 NOE-based distance restraints. The backbone (N-C-C = O) RMSD from the average structure of an ensemble of 15 out of 20 calculated structures was 0.54 +/- 0.16 A. Apart from some dynamic fraying at both termini, the distance geometry and simulated annealing calculations showed that apoA-II(18-30)+ adopted a well defined amphipathic helix with distinct hydrophobic and hydrophilic faces.

Conformation of human serum apolipoprotein A-I(166-185) in the presence of sodium dodecyl sulfate or dodecylphosphocholine by 1H-NMR and CD. Evidence for specific peptide-SDS interactions.

The segment, YSDELRQRLAARLEALKENG, corresponding to residues 166 to 185 of human serum apolipoprotein A-I, was studied by circular dichroism and NMR spectroscopy in sodium dodecyl sulfate and dodecylphosphocholine micelles. 2-Dimensional NOESY, TOCSY and DQF-COSY spectra of apoA-I(166-185) in perdeuterated sodium dodecyl sulfate (SDS-d25) and dodecylphosphocholine (DPC-d38) micelles were collected at a peptide/SDS (DPC) ratio of 1:40. Similar CD spectra and NOE connectivity patterns were observed for apoA-I(166-185) in SDS and DPC, indicating a similar helical conformation in both. Conformations of apoA-I(166-185) in DPC-d38 micelles, and in SDS-d25 micelles at two pH values, 6.6 and 3.7, were determined using distance geometry calculations. Backbone superposition (N,C alpha,C = O) for an ensemble of twenty-nine structures in DPC at pH 6.0 gave a RMSD of 0.45 +/- 0.09 A for the region D168 to K182, while for all atoms it was 1.60 +/- 0.17 A. In SDS, the ensemble of nineteen structures each at pH 6.6 and 3.7 gave RMSDs of 0.28 +/- 0.07 A and 0.35 +/- 0.10 A, respectively, for the region D168 to K182. RMSD for superposition of all atoms was 1.36 +/- 0.10 A and 1.38 +/- 0.21 A at the respective pH values. In all cases a highly defined class A amphipathic helical structure was found for the region R171 to K182. Since the same structure occurs in micelles with either negatively charged or zwitterionic head groups it strongly suggests a dominant role for hydrophobic interactions in stabilizing the complex. The Y166 aromatic ring is bent back upon the helix axis at the lower pH. NMR determination of pKa values for D168, E169, E179 and E183 in the presence of SDS or DPC indicated a micro-pH at the micellar surface approximately one pH unit higher than the normal residue pKa. SDS interactions with the peptide were examined by collecting 1H NOESY spectra in the presence of protiated SDS. Residues R171, R173, R177, as well as the aromatic ring of Y166, were shown by intermolecular NOE measurements to interact with SDS, hence a key interaction in stabilizing the complex appears to be between interfacial basic side-chains and SDS alkyl chains.

Structural studies of a peptide activator of human lecithin-cholesterol acyltransferase.

The synthetic lipid-associating peptide, LAP-20 (VSSLLSSLKEYWSSLKESFS), activates lecithin-cholesterol acyltransferase (LCAT) despite its lack of sequence homology to apolipoprotein A-I, the primary in vivo activator of LCAT. Using SDS and dodecylphosphocholine (DPC) to model the lipoprotein environment, the structural features responsible for LAP-20's ability to activate LCAT were studied by optical and two-dimensional 1H NMR spectroscopy. A large blue shift in the intrinsic fluorescence of LAP-20 with the addition of detergent suggested that the peptide formed a complex with the micelles. Analysis of the CD data shows that LAP-20 lacks well defined structure in aqueous solution but adopts helical, ordered conformations upon the addition of SDS or DPC. The helical nature of the peptides in the presence of both lipids was confirmed by upfield H alpha NMR secondary shifts relative to random coil values. Average structures for both peptides in aqueous solutions containing SDS and DPC were generated using distance geometry methods from 329 (SDS) and 309 (DPC) nuclear Overhauser effect-based distance restraints. The backbone (N, Calpha, C=O) RMSD from the average structure of an ensemble of 17 out of 20 calculated structures was 0.41 +/- 0.15 Angstrom for LAP-20 in SDS and 0.41 +/- 0.12 A for an ensemble of 20 out of 20 calculated structures for LAP-20 in DPC. In the presence of SDS, the distance geometry and simulated annealing calculations show that LAP-20 adopts a well defined class A amphipathic helix with distinct hydrophobic and hydrophilic faces. A similar structure was obtained for LAP-20 in the presence of DPC, suggesting that both detergents may be used interchangeably to model the lipoprotein environment. Conformational features of the calculated structures for LAP-20 are discussed relative to models for apolipoprotein A-I activation of LCAT.

Conformation of two peptides corresponding to human apolipoprotein C-I residues 7-24 and 35-53 in the presence of sodium dodecyl sulfate by CD and NMR spectroscopy.

Peptides corresponding to the proposed lipid-binding domains of human apolipoprotein C-I, residues 7-24 (ALDKLKEFGNTLEDKARE) and 35-53 (SAKMREWFSETFQKVKEKL), were studied by CD and two-dimensional 1H NMR spectroscopy. Sodium dodecyl sulfate (SDS) was used to model the lipoprotein environment. Analysis of the CD data shows that both peptides lack well-defined structure in aqueous solution but adopt helical, ordered structures upon the addition of SDS. The helical nature of the peptides in the presence of SDS was confirmed by H alpha secondary shifts. A total of 199 (apoC-I(7-24)) and 266 (apoC-I(35-53)) distance restraints were used in distance geometry and simulated annealing calculations to generate average structures for both peptides in aqueous solutions containing SDS. The backbone (N, C alpha, C = O) RMSD from the average structure of an ensemble of 20 structures was 0.73 +/- 0.22 and 0.48 +/- 0.14 A for apoC-I(7-24) and apoC-I(35-53), respectively. In the presence of SDS, the distance geometry and simulated annealing calculations show that both peptides adopt well-defined amphipathic helices with distinct hydrophobic and hydrophilic faces. The calculated structures are discussed relative to predicted structures. Comparing our CD and NMR results for the apoC-I fragments in SDS with CD results of others obtained in the presence of dimyristoylphosphatidylcholine indicates that SDS may be a better model of the lipoprotein environment.

Sequence-specific 1H NMR assignments and secondary structure of a lipid-associating peptide from human ApoC-I: an NMR study of an amphipathic helix motif.

The conformation of a synthetic peptide corresponding to residues 35-53 (SAKM-REWFSETFQKVKEKL) of human apolipoprotein C-I (57 amino acids) was studied by nuclear magnetic resonance and circular dichroism spectroscopy in water and in perdeuterated dodecylphosphocholine solution at 37 degrees C and pH 4.8. The proton resonances of the peptide in both solutions were assigned from TOCSY, NOESY and DQF-COSY experiments. In water solution, the peptide is predominantly "random", although nuclear Overhauser connectivity patterns and H alpha secondary shifts show a threshold population of nascent helical conformers. Upon the addition of 40-fold molar excess dodecylphosphocholine to the water solution, the peptide adopts a helical structure that extends throughout the sequence.

Secondary structures of lipid-associating peptides: a Fourier transform infrared study.

Four peptides from 20 to 28 residues in length were studied by Fourier transform infrared (FTIR) spectroscopy in solution and in complexes with dimyristoylphosphatidylcholine (DMPC). The four peptides included the 20-residue lipid-associating peptide, LAP-20, which was predicted to form an amphipathic helical structure in the presence of lipids, and three other peptides whose sequences had less amphipathic helix-forming properties. The complexes were shown by electron microscopy to be discoidal in shape with mean diameters of 21-27 nm. At the concentrations used for IR, the peptides appeared to form oligomers consisting of intermolecular beta-sheets. In the presence of lipids, the amount of beta-structure decreased; however, amounts of beta-structure were still approximately equal to amounts of alpha-helix. The IR results for LAP-20 contradicted previous circular dichroism results that predicted 50%-90% alpha-helix in DMPC complexes. Convex constraint analysis (CCA) deconvolution of the circular dicroism (CD) spectrum to estimate secondary structures predicted amounts of helix similar to those predicted by IR, but there was still substantial disagreement between IR and CD estimates of other secondary structures. For LAP-20 in complexes, CD predicted random structure. Possible physiological consequences of partial disordering of peptide structures are discussed.

An alternate apoprotein conformation in high density apolipoprotein discoidal complexes. A Fourier transform infra-red study.

Discoidal complexes have been prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the apoproteins of HDL3 (apo HDL3) or purified apo A-I. Gel electrophoresis established that apo HDL3 contained 74% apo A-I. Deconvolution and curve-fitting of the infra-red amide I band of the apoprotein in the lipid-protein complex revealed a secondary structure containing approximately 40% alpha-helix and 50% beta-structure. This contrasted with the results from circular dichroism studies (Surewicz et al. (1986) J. Biol. Chem., 261, 16191) of apo A-I/DMPC complexes which predicted 68% alpha-helix and 7% beta-structure. The discrepancy between the two methods and limitations of the two techniques for lipoproteins is discussed.

Deuterium NMR study of the interaction of phytanic acid and phytol with the head group region of a phospholipid bilayer. Evidence of magnetic orientation.

Deuterium nuclear magnetic resonance (2H-NMR) spectra of multilamellar dispersions of 1,2-dipalmitoyl-sn-glycero-3-phospho-[1',2'-methylene-2H4]choline (DPPC-d4) containing 20 mol% of the isoprenoid compounds phytol or phytanic acid in excess deuterium-depleted water, or Tris buffer (pH 7.4), have been recorded over the temperature range 15-55 degrees C. Phytol (20 mol%) causes a decrease in the residual quadrupolar splitting, delta vQ, of the choline 1'-deuterons (i.e., those adjacent to the phosphate moiety) by 3%, while delta vQ, of the 2'-deuterons decrease by 22%. Phytanic acid (20 mol%) reduces delta vQ of the 2'-deuterons by 43% and increases delta vQ of the 1'-deuterons by approx. 16%. First (M1) and second (M2) moments were calculated from the spectra. Plots of M1 and M2 versus temperature suggest phytol and phytanic acid have a significant effect on the pretransition. Spin-lattice relaxation times (T1) and transverse relaxation times (T2e, the time constant for decay of the quadrupolar echo) have been determined over the temperature range 15-50 degrees C. The presence of 20 mol% phytanic acid results in orientation of the DPPC bilayers in the magnetic field with the phospholipid long axes oriented perpendicular to the magnetic field as shown by 2H- and 31P-NMR. The sample must be carried through several freeze-thaw cycles in the presence of Tris buffer in order that the phospholipid magnetic field ordering occur. Possible explanations for the effect of phytol and phytanic acid on the dynamic structure of the choline head group of phospholipids in the bilayer are suggested.

Structure and motion of phospholipids in human plasma lipoproteins. A 31P NMR study.

The structure and motion of phospholipids in human plasma lipoproteins have been studied by using 31P NMR. Lateral diffusion coefficients, DT, obtained from the viscosity dependence of the 31P NMR line widths, were obtained for very low density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoproteins (HDL2, HDL3), and egg PC/TO microemulsions at 25 degrees C, for VLDL at 40 degrees C, and for LDL at 45 degrees C. At 25 degrees C, the rate of lateral diffusion in LDL (DT = 1.4 x 10(-9) cm2/s) is an order of magnitude slower than in the HDLs (DT = 2 x 10(-8) cm2/s). At 45 degrees C, DT for LDL increases to 1.1 x 10(-8) cm2/s. In contrast, DT for VLDL increases only slightly going from 25 to 40 degrees C. The large increase in diffusion rate observed in LDL occurs over the same temperature range as the smectic to disordered phase transition of the core cholesteryl esters, and provides evidence for direct interactions between the monolayer and core. In order to prove the orientation and/or order of the phospholipid head-group, estimates of the residual chemical shift anistropy, delta sigma, have been obtained for all the lipoproteins and the microemulsions from the viscosity and field dependence of the 31P NMR line widths. For VLDL and LDL, the anisotropy is 47-50 ppm at 25 degrees C, in agreement with data from phospholipid bilayers. For the HDLs, however, significantly larger values of 69-75 ppm (HDL2) and greater than 120 ppm (HDL3) were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Insoluble complex formation between low density lipoprotein and heparin. A 31P-NMR study.

31P-NMR has been used to probe the motions of the phosphate moiety of phospholipid head-groups in samples of human low density lipoprotein (LDL) in which particle tumbling has been greatly reduced by increasing the viscosity of the medium, by forming an LDL gel by ultracentrifugation, or by precipitation with heparin. The 31P-NMR spectra of LDL gel give broad "powder-like" lineshapes, with the sign and magnitude of the anisotropy characteristic of the bilayer mesophase, which narrow as the temperature is raised from 5 to 45 degrees C. This narrowing occurs over the same temperature range as the core cholesteryl ester liquid-crystalline to liquid phase transition, suggesting interactions between the surface and core. The 31P lineshapes of LDL-heparin insoluble complexes are also "powder-like", but are broader than native LDL at all temperatures studied. The spectra were simulated assuming an axially-symmetric shielding tensor motionally narrowed by Brownian isotropic diffusion [Burnell et al. (1980) Biochim. Biophys. Acta 603, 63-69], allowing determination of the lateral diffusion coefficients, DT, and the chemical shift anisotropy, delta sigma, of the monolayer phospholipids. Relative to LDL gel, the temperature-dependence of DT was reduced in the LDL-heparin insoluble complexes, and delta sigma was increased from 50 to 60 ppm. The results suggest that insoluble complex formation slows phospholipid lateral diffusion in the LDL monolayer and alters the orientation and/or order of the head-group.