Studies on mixed micelles of triton X–100 and phosphatidylcholine using nuclear magnetic resonance techniques

The addition of the nonionic detergent Triton X-100 to aqueous phosphatidyl-choline dispersions converts the bilayer structures to mixed micellar structures containing Triton X-100. High-resolution nuclear magnetic resonance spectroscopy at 220 MHz was used to follow this conversion, and the general spectral characteristics of the mixed micelles are presented. The results are discussed in terms of the precise change in structure which occurs as Triton is mixed with the phospholipid bilayers, and it is concluded that, above a molar ratio of about 2:1 Triton to phospholipid, most or all of the phospholipid is in mixed micelles. The relevance of these results to the study of enzymes which require substrate in the form of micelles is discussed.     

500 MHz H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

13C nuclear magnetic resonance studies of egg phosphatidylcholine.

Spin lattice relaxation times (T1) and apparent spin-spin relaxation times (T2) derived from linewidth have been used to investigate model membranes composed of egg yolk phosphatidylcholine. T1 measurements appear to be largely dominated by segmental motion and as a consequence are not very sensitive to small changes in membrane structure. On the contrary, apparent T2 times are shown to be sensitive to such changes in the membrane and are thus suggested as a useful tool for further investigation of membrane structure.     

500 MHz 1H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

Deuterium magnetic resonance studies of phospholipid bilayers

L-α-dipalmitoyl lecithin is selectively deuterated at two different chain positions. The residual quadrupole splittings of the corresponding phospholipid bilayers are measured by means of deuterium magnetic resonance and evaluated in terms of the segmental order parameters. The results are briefly compared with other esr and nmr investigations of lipid bilayers.     

Structure and dynamics of melittin in lysomyristoyl phosphatidylcholine micelles determined by nuclear magnetic resonance.

Mixed micelles of the 26-residue, lytic peptide melittin (MLT) and 1-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) in aqueous solution at 25 degrees C were investigated by (13)C- and (31)P-NMR spectroscopy. (13)C alpha chemical shifts of isotopically labeled synthetic MLT revealed that MLT in the micelle is predominantly alpha-helical and that the peptide secondary structure is stable from pH 4 to pH 11. Although the helical transformation of MLT as determined from NMR is evident at lipid:peptide molar ratios as low as 1:2, tryptophan fluorescence measurements demonstrate that well-defined micellar complexes do not predominate until lipid:peptide ratios exceed 30:1. (31)P linewidth measurements indicate that the interaction between phosphate ions in solution and cationic groups on MLT is pH dependent, and that the phosphoryl group of MMPC senses a constant charge, most likely +2, on MLT from pH 4 to pH 10. (13)C-NMR relaxation data, analyzed using the model-free formalism, show that the peptide backbone of MLT is partially, but not completely, immobilized in the mixed micelles. Specifically, order parameters (S(2)) of C alpha-H vectors averaged 0.7 and were somewhat larger for residues in the N-terminal half of the molecule. The amino terminal glycine had essentially the same range of motion as the backbone carbons. Likewise, order parameters for the trp side chain were similar to those found for the peptide C alpha moieties, as was verified by trp fluorescence anisotropy decay data. In contrast, the motion of the lysine side chains was less restricted, the average S(2) values for the C epsilon-H vectors being 0.19, 0.30, and 0.44 for lys-7, 21, and 23, respectively, for MLT in the mixed micelles. Values of the effective correlation time of the local motion tau e were in the motional narrowing limit and usually longer for side-chain atoms than for those in the backbone. The dynamics were independent of pH from pH 4 to pH 9, but at pH 11 the correlation time for the rotational motion of the mixed micelles as a whole increased from 10 ns to 16 ns, and S(2) for the lys side chains increased. Overall it appears that the MLT helix lies near the surface of the micelle at low to neutral pH, but at higher pH its orientation changes, accompanied by deeper penetration of the lysine side chains into the micelle interior. It is apparent, however, that the MLT-lipid interaction is not dependent on deprotonation of any of the titratable cationic groups in the peptide in the pH 4-10 range, and that there is substantial backbone and side-chain mobility in micelle-bound MLT.     

Structural and dynamical studies of mixed chlorophyll/ phosphatidylcholine bilayers via x-ray diffraction,absorption polarization spectroscopy and nuclear magnetic resonance

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidtlcholine fatty acid chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a $\text{10}{}^{\mathrm{?9}}\text{? 10}{}^{\mathrm{?10}}\text{s}$ time scale in the ideally mixed two-component bilayer.     

Proton magnetic relaxation studies of mixed phosphatidylcholine/fatty acid and mixed phosphatidylcholine bimolecular bilayers.

High resolution proton spin-lattice relaxation times (T1), spin-spin relaxation times (T2) and resonance linewidths were measured above the gel-to-liquid crystal transition temperature (Tm), in phosphatidylcholine bilayers possessing various degrees of intramolecular motional anisotrophy at the level of various alkyl chain proton groups. The experiments were designed to test the hypothesis that coupled trans-gauche isomerizations along the chains can be responsible for the anisotropic motion of phosphatidylcholine proton groups in bilayer membranes (Horwitz, A.F., Horsley, W.J. and Klein, M.O. (1972) Proc. Natl. Acad. Sci. U.S. 69,500). Systematic series of structural perturbations of the bilayer were achieved in mixed phosphatidylcholine/fatty acid and in mixed phosphatidylcholine bilayers where the degree of motional anisotrophy of the chains' proton groups was gradually reduced by progressively increasing the chain length disparity of the two components. The systematic T1 and T2 variations observed were interterpreted on the basis of the Woessner's treatment for computing the relaxation times of a spin pair reorienting randomly about an axis which, in turn, tumbles randomly (Woessner, D.E. (1962) J. Chem. Phys. 36, 1). The results confirmed in a qualitative sense the original hypothesis made by Horwitz et al. The time-averaged structural interpretations suggested by the mangetic relaxation studies are in agreement with low-angle X-ray diffraction results obtained below Tm. In addition, the T1 values evaluated at various temperatures in dipalmitoyl phosphatidylcholine vesicles incorporated with either 2H-labeled or unlabeled palmitic acid chains indicated that the average intermolecular contribution to the spin-lattice relaxation rate of the proton groups of the phosphatidylcholine chains appears comparable to the intramolecular term at temperatures moderately higher than Tm, but becomes less and less important as the temperature is further increased above the thermal transition.     

Structural and dynamical studies of mixed chlorophyll/phosphatidylcholine bilayers via x-ray diffraction, absorption polarization spectroscopy and nuclear magnetic resonance.

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidylcholine fatty acids chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a 10(-9)-10(-10)s time scale in the ideally mixed two-component bilayer.     

Deuterium magnetic resonance of selectively deuterated cholesteryl esters in dipalmitoyl phosphatidylcholine dispersions

Dispersions (50 wt% water) containing 95 mol% dipalmitoyl phosphatidylcholine/5 mol% deuterated cholesteryl palmitate (or stearate) were studied using ²H-NMR. Incorporation of ester into the phospholipid bilayer was found to be 0.5 mol% at 50°C. From the profile of ²H quadrupolar splitting vs. chain position, support for an average conformation resembling a ‘horseshoe’ within the bilayer is obtained. Quadrupolar relaxation times $\text{T}{}_{2e}$ of approx. 250 μs and approx. 850 μs are measured for cholesteryl palmitate-2,2-d₂ and cholesteryl palmitate-16,16,16-d₃, respectively, which are less than one-half those obtained for the corresponding positions in dipalmitoyl-d₆₂ phosphatidylcholine. This is ascribed to a slower rate of motion of the ester chain and/or an extra, slow motion of the molecule.     

Proton nuclear magnetic resonance studies on the molecular dynamics of plasmenylcholine/cholesterol and phosphatidylcholine/cholesterol bilayers

Physiologically relevant molecular species of plasmenylcholine and phosphatidylcholine were synthesized and their molecular dynamics and interactions with cholesterol were compared by determination of salient proton spin-lattice relaxation times and apparent activation energies for 1H-NMR observable motion. The molecular dynamics of PA PhosCho (1-hexadecanoyl-2-eicosatetra-5',8',11',14'-enoyl-sn-glycero-3-pho sphocholine) in multiple regions of the bilayer. Furthermore, the fluidity gradient of PA PhosCho was larger than that of PA PlasCho as ascertained by 1H spin-lattice relaxation time measurements. Introduction of cholesterol into each bilayer resulted in disparate effects on the dynamics of each subclass including: (1) increased motional freedom in the polar head group of PA PlasCho without substantial alterations in the dynamics of the polar head group of PA PhosCho; and (2) increased immobilization of the membrane interior in PA PlasCho in comparison to PA PhosCho. Analysis of Arrhenius plots of T1 relaxation times demonstrated that the apparent activation energies for vinyl and bisallylic methylene proton NMR observable motion in PA PhosCho were greater than that in PA PlasCho. Thus, comparisons of spin-lattice relaxation times and apparent activation energies demonstrate that vesicles comprised of PA PlasCho and PA PhosCho possess differential molecular dynamics and distinct interactions with cholesterol. Collectively, these results underscore the significance of the conjoint presence of the vinyl ether linkage and arachidonic acid as an important determinant of membrane dynamics in specialized mammalian membranes.     

Deuterium nuclear magnetic resonance investigation of dimyristoyllecithin--dipalmitoyllecithin and dimyristoyllecithin--cholesterol mixtures

Deuterium nuclear magnetic resonance (NMR) spectra of 1,2-dimyristoyl-3-sn-phosphatidylcholines (DMPCs) specifically deuterated in the 2-chain at one of positions 2', 3', 6', or 14' have been obtained by the quadrupole-echo Fourier transform method at 34.1 MHz (corresponding to a magnetic field strength of 5.2 T) or the pure material as a function of temperature, and in the presence of either 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) or cholesterol as a function of temperature and composition. The results with pure DMPC and DMPC--DPPC mixtures indicate that a sharp, intense deuterium resonance is characteristic of fluid-phase lipids, whereas a broad resonance is characteristic of solid-phase lipids. There is shown to be good agreement between the deuterium NMR derived DMPC--DPPC phase diagram and that derived by using other techniques. The deuterium NMR results obtained with the DMPC--cholesterol system are not interpreted in terms of a phase diagram. They do indicate, however, that the transition breadth is increased considerably and the temperature at which the lipid chains "solidify" is depressed by the addition of cholesterol to the DMPC bilayer. The particular nature of the increase and the depression is found to be dependent on where the label is located on the lipid.     

Deuterium and phosphorus nuclear magnetic resonance studies on the binding of polymyxin B to lipid bilayer-water interfaces

Deuterium and phosphorus NMR methods have been used to study the binding of polymyxin B to the surface of bilayers containing lipids that were deuterated at specific positions in the polar head-group region. The binding of polymyxin B to acidic dimyristoylphosphatidylglycerol (DMPG) membranes induces only small structural distortions of the glycerol head group. The deuterium spin-lattice relaxation times for the different carbon-deuterium bonds in the head group of the same phospholipid are greatly reduced on binding of polymyxin B, indicating a restriction of the motional rate of the glycerol head group. Only very weak interactions were detected between polymyxin B and bilayers of zwitterionic dimyristoylphosphatidylcholine (DMPC). In mixed bilayers of the two phospholipid types, in which either of the two phospholipids was deuterated, the presence of polymyxin B caused a lateral phase separation into DMPG-enriched phospholipid-peptide clusters and a DMPG-depleted phase. Complete phase separation did not occur: peptide-containing complexes with charged phosphatidylglycerol contained substantial amounts of zwitterionic phosphatidylcholine. Exchange of both phospholipid types between complexes and the bulk lipid matrix was shown to be fast on the NMR time scale, with a lifetime for phospholipid-peptide association of less than 1 ms.     

Deuterium magnetic resonance study of the gel and liquid crystalline phases of dipalmitoyl phosphatidylcholine.

Deuterium magnetic resonance is applied to the study of the liquid crystalline and gel phases, and of the phase transition, of a multilamellar dispersion of chain perdeuterated (d62)-dipalmitoyl phosphatidylcholine/H2O. Analysis of the deuterium spectra in terms of the moments of the spectra allows one to make quantitative statements concerning the distribution of quadrupolar splittings even in complicated situations, e.g., when using perdeuterated sampled or when there are mixed phases. This analysis indicates that d62-dipalmitoyl phosphatidylcholine in excess H2O undergoes a sharp phase transition (with a width of less than 1 degree C) at approximately 37 degrees C and that there appears to be hysteresis in the phase transition of approximately 1 degree C. In the lamellar liquid crystalline phase above 37 degrees C the spectra show a number of well-resolved features whose quadrupolar splittings can be followed as the temperature is varied. The gel phase near 20 degrees C possesses a very broad, almost featureless spectrum that does not seem to support a model of the gel phase wherein the hydrocarbon chains are fully extended in the all-trans conformation. At temperatures near 0 degrees C the spectra clearly indicate that a large fraction of the lipid molecules cease the rotation about their long axes, giving a spectrum more characteristic of a rigid or solid sample. These results give a picture of the gel phase as a phase characterized by considerable hydrocarbon chain disorder near 20 degrees C and becoming a more solid-like phase near 0 degrees C. The spin-lattice relaxation time, T1, has been measured at 20 degrees C in the gel phase, and at 37 and 45 degrees C in the liquid crystalline phase. The values of T1 obtained for each of the resolvable peaks in the spectrum at 37 degrees C are compared to the values (for each peak) of T2e, the decay time of the quadrupolar echo, obtained at the same temperature. These results are discussed in terms of a simple two-motion model.     

High-resolution nuclear magnetic resonance studies of proteins

The combination of advanced high-resolution nuclear magnetic resonance (NMR) techniques with high-pressure capability represents a powerful experimental tool in studies of protein folding. This review is organized as follows: after a general introduction of high-pressure, high-resolution NMR spectroscopy of proteins, the experimental part deals with instrumentation. The main section of the review is devoted to NMR studies of reversible pressure unfolding of proteins with special emphasis on pressure-assisted cold denaturation and the detection of folding intermediates. Recent studies investigating local perturbations in proteins and the experiments following the effects of point mutations on pressure stability of proteins are also discussed. Ribonuclease A, lysozyme, ubiquitin, apomyoglobin, alpha-lactalbumin and troponin C were the model proteins investigated.