We report here the results of 2H solid state NMR studies on water present in the interbilayer space of the phosphatidyl choline bilayers. Mechanically oriented DMPC bilayers were prepared by alligning the sample in between glass plates and the sample was fully hydrated with D2O to observe the behavior of water in between the bilayers. 2H NMR spectra were recorded both above and below the gel to liquid crystalline phase transition temperatures as well as sub-zero temperatures. Spectra above the gel to liquid crystalline phase transition temperature show a doublet apart from an isotropic line and the splitting of the doublet increases with increasing temperature. The doublet structure merges with the isotropic signal into a single line near the phase transition temperature. Spectra below the transition temperature show the single line splitting into a doublet once again and the splitting of this doublet increases with lowering temperature. Around 0 degrees C we can see at least two doublet structures developing. As the temperature is further lowered the individual lines broaden and join into a single line around -40 degrees C. There are indications for possible further splitting to appear in the spectrum around -70 degrees C, which we are in the process of verifying. Angular dependence and relaxation studies are further underway on this and other PC systems using the oriented sample technique to understand the conformation of the interbilayer water molecules, especially at low temperatures where the motional processes are expected to slow down considerably.
The synthesis and mesomorphic properties of the liquid crystals, 4,4'-di-n-alkyldiphenyldiacetylenes are described. These highly symmetrical hydrocarbon liquid crystal compounds are of interest because they are nonpolar and have a ideally linear mesogenic core shape. These attributes make these compounds interesting for NMR studies of their phase behavior and solute-solvent interactions. They are expected to simple depend on excluded volume interactions associated with the shapes of the solute-solvent pair. The neat thermotropic liquid crystals are studied by the carbon-13 separated local field spectroscopy combined with variable angle spinning (SLF/VAS) technique. The solute-solvent interaction is derived from analysis of incompletely averaged dipolar and quadrupolar interactions in the spectra of the solute molecules.
Field-oriented phospholipid bilayer disks, or bicelles, can mimic the environment of biological membranes while allowing unique spectroscopic measurements of structural and dynamic properties. In NMR, dipolar couplings and CSA data for various magnetic isotopes yield information on conformational and motional properties of bilayer associated molecules. Moreover, these parameters can be measured with adequate resolution to work with multiple isotope labels in single molecular entities. These methods have been successfully used in studies of membrane-bound carbohydrates. The applicability to the study of surface-associated peptides and proteins is now being explored. Site specifically 13C and 15N labeled peptides were synthesized with and without a lipid anchor based on the sequence of the N-terminal amphipathic helix of ARF1, a myristoylated protein important for membrane trafficking. The dipolar couplings and CSA data acquired by carbon and nitrogen observe NMR experiments are used to study the orientation of the helix with respect to the bilayer normal and to compare the structure and dynamic behavior of the anchored and the surface-associated peptide on the membrane surface.
Stimulated echo pulsed field gradient methods are used to study the diffusion of unfrozen water in frozen porous media. Problems related to pfg eddy currents, short T2, and temperature gradients are dealt with through modification of existing pulse sequences used for this purpose. The data are analyzed in terms of discrete and continuous distributions of diffusion coefficients. Results indicate that while the unfrozen water content in these systems is extremely temperature dependent around the freezing point, the molecular diffusivity of this water is not.
The gel electrolyte systems are composed of solvent, salt and polymer and form inhomogeneous structures. Generally, the gels are intermediate between solid and liquid and the internal fluidity has particular significance for their application as gel electrolytes. Consequently, direct measurement of the translational diffusion of the components is very useful for understanding the gel structures. We have measured the translation diffusion coefficients of all the components of the gel electrolyte. The diffusion of the lithium ions and anions was measured using 7Li and 19F pulsed field gradient (PFG) NMR, respectively, and the solvents and the matrix polymer were measured using 1H PFG NMR. Comparison were made between the gamma-butyloractone(GBL) - LiBF4 - cross-linked polyethyleneoxide (PEO) and propylenecarbonate (PC) - LiN(SO2CF3)2 - cross-linked PEO systems. In the former system all the species diffuse freely, but in the latter system the diffusion of the lithium ion is some times restricted. In these systems the order of the diffusion coefficients (fastest to slowest) is solvent, anion, lithium ion and polymer. The ion solvation effect of GBL is much more pronounced than in the PC system. The different solvation potential results in different gel structures in the GBL and PC systems. The diffusion coefficients of the individual components in the gels demonstrate that the gel structures allows deeper insight into the gel structure, transport phenomena and conductivity.
In the general case of isotropic rotational diffusion, internuclear dipolar couplings average to zero. However, partial alignment of solute molecules with the magnetic field may be achieved in aqueous mixtures of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). As a result, dipolar couplings between pairs of 1H, 13C or 15N nuclei are no longer averaged to zero and they can be readily measured. These residual dipolar couplings provide valuable structural information promising to improve the accuracy and precision of NMR structures in solution [1,2]. In this poster we present the results of a detailed characterization of DMPC/DHPC mixtures. Such mixtures may adopt an ordered, dilute liquid crystalline phase of disc-shaped bicelles. However, the stability of these liquid crystals and the degree of induced alignment in the solute molecules depend strongly on experimental conditions such as composition and concentration of the DMPC/DHPC mixtures, temperature, pH and salt concentration. Based on these measurements we propose optimal conditions for sample preparation and storage. In addition, we present measurements of residual dipolar couplings and discuss some interesting implications. [1] A. Bax and N. Tjandra, J. Biomol. NMR., 10, 289 (1997). [2] N. Tjandra and A. Bax, Science, 278, 1111 (1997).
Detailed molecular information on an atomic level about the nature and specificity of the interactions of peripheral proteins with membranes is not widely available. However, 31-P MAS NMR enabled us to study the properties of each individual phospholipid component in complex membranes upon binding of peripheral proteins and to monitor specific interactions and domain formation. Membrane association of a number of important peripheral proteins (e.g. Src, MARCKS HIV-1 Gag, K-Ras) requires nonspecific electrostatic interactions between a cluster of basic residues on the protein and acidic phospholipids in the membrane. MARCKS peptide (effector region of Myristoylated Alanine-Rich C Kinase Substrat) has been shown do produce lateral domain formation. Using 31 P MAS NMR we could directly monitor domain formation introduced by pentalysine, which correspond to the first five residues of MARCKS peptide. We also showed that the interactions are purely electrostatic, thereby segregating the acidic lipid out of mixed membranes into a new spectroscopically observable domain. Sideband analysis enabled us to quantify changes in the properties for each lipid component in the different domains, something not possible with most other techniques. Myosin, an important biological motor protein, was also studied by 31P MAS NMR. It could be shown that the protein interacts specifically in an ATP-dependent way with the charged lipid component in mixed bilayers; a mechanism important for its biological function.
The Iron Responsive Element RNA hairpin has a six-nucleotide loop with an A:U loop closing base pair. A single noncanonical interaction involving the first (C1) and fifth (G5) nucleotide of the loop provides some structure to the loop, but the fourth (U4) and sixth (C6) nucleotides are unconstrained by the NMR data while the second (A2) and third (G3) are stacked above C1. The first G:C pair of the stem is also unstable, and shows several resonances, indicating slow exchange on the NMR timescale. Above 30C, there is evidence of increased flexibility of the loop nucleotides, suggesting that the loop structure is not tightly constrained. This RNA is used in 13C relaxation experiments to determine the dynamics of the nucleotide bases in the loop and in the stem. The C8 and C2 positions of the purine bases, with their attached 1H, are isolated spin pairs, so relaxation of the 13C is dominated by dipolar and CSA contributions without complications from homonuclear cross-correlation. 13C-Adenosine and/or 13C-Guanosine labelled RNAs are used in heteronuclear relaxation experiments and the data analyzed by the Lipari & Szabo model-independent formalism to obtain tumbling times, order parameters, and exchange rates of the bases in the loop and the stem. The temperature-dependence of the dynamics parameters show a clear pattern of increased motions in the RNA loop.
1H-13C heteronuclear dipolar correlation 2-D NMR spectroscopy (2-D HETDIPCOR) has been used to investigate the interaction of [4-13C]-cholesterol with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in artificial membranes (3/7 sterol to phospholipid mol ratio) in excess water at 15-45 C (liquid crystalline phase). By varying the mixing (cross-polarization) time, the 2D NMR specra permit identification of the different 1H nuclei that contribute to the polarization of the labeled 13C site. These include the directly attatched protons as well as those bound to the neighboring carbon atoms (intramolecular cross-polarization), plus a resonance at 4.55 ppm, identified as the proton attatched to the C1 carbon of the glycerol moiety of DMPC (intermolecular cross-polarization). The same spectral characteristics were observed at 15 C, showing the stability of the liquid ordered (lo) DMPC/cholesterol phase through the phase transition of the phospholipid matrix. Similar studies on [4-13C]-cholesterol/bovine brain sphingomyelin (3/7 mol ratio) at 35 C identified a resonance at 4.3 ppm in the sphingolipid contributing to the polarization of the labeled carbon in the sterol. This site was identified as the proton attatched to the C3 carbon of the sphingosine moiety. Furthermore, it was found that the intermolecular cross-polarization peak was not present in [4-13C]-cholesterol/ POPE membranes at the same molar ratio and 35 C, indicating a close positioning between cholesterol and phosphatidylcholine or sphingomyelin, in agreement with previous studies. Finally, 2D-HETDIPCOR spectra of biosynthetically 13C- labeled ergosterol (65% labeled at positions 2,4,6,8,10,11,12,14,16,20, and 23) incorporated in POPC (3/7 mol ratio) were obtained at 25 C, which allowed mapping of the interactions of this sterol and its phospholipid matrix. Taken together these results indicate that 2D-HETDIPCOR can provide detailed information on intermolecular interactions in lipid membranes under physiological conditions.
Reverse micelle (RM) solutions of NiCl2(aq), decane, and the surfactant bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) have recently been proposed as the proton NMR signal component in an MRI phantom. Characterization of the T1 values of the components of the RM solution allows for optimization of the solution for use as a phantom and also provides insight into the rotational dynamics of individual water and decane molecules, and of the reverse micelles themselves. Proton T1 values have been measured for the water, decane-CH2, and decane-CH3 NMR signals from NiCl2(aq)-decane-AOT mixtures having phi, defined as volume(water+AOT)/total volume, ranging from 0-0.95, and temperature, T, ranging from 0-40 degrees C. The phi range includes both the RM phase and a lamellar phase. T1 shows markedly different trends in the two phases. For the RM phase, as T decreases and phi increases, the CH2 and CH3 T1 decreases, which can be shown to indicate decreasing rotational motions of the two groups (at the Larmour frequency = 300 MHz). These results are consistent with the decane molecules becoming more restricted between neighboring reverse micelles as phi increases, and less energetic as T decreases. The water T1 increases as T decreases, and decreases only slightly as phi increases. The temperature dependence can be rationalized as before, and the near phi independence shows that the reverse micelles act almost independently of each other, with the small decrease in T1 perhaps indicating the effect of reverse micelle percolation. In the high phi lamellar phase, the decane T1 is independent of T and decreases as phi increases, showing that the packing of the decane molecules allows freer motion with an increase in decane layer thickness, but not with the increase in T. The water T1 data suggest that the microscopic environment in the water layers is similar to that in the reverse micelles. Measurement of the self-diffusion coefficients of the water, decane, and AOT molecules in these solutions is currently in progress.
Polypeptides that are immobilized by strong interactions with phospholipid bilayers can be studied by means of solid-state NMR spectroscopy. In uniaxially aligned systems the anisotropy and the size of orientationally dependent nuclear interactions is revealed using these methods. This information can be used to analyse the structure, dynamics and topology of bilayer-associated polypeptides. Solid-state NMR measurements have been used in the past to calculate the secondary structure and orientation of synthetic polypeptides in oriented lipid bilayers. Novel NMR techniques which are based on the line narrowing of phase and frequency switched Lee-Goldburg proton decoupling allow one to resolve approximately 200 resonances from the 15N uniformly labeled thermolytic fragments of colicin B. The high resolution of these two-dimensional PISEMA spectra promises that the complete structure determination of membrane proteins will be possible once assignment strategies have been developed. Approximately 50 amide residues of the colicin B channel fragment resonate at chemical shifts > 180 ppm which is in agreement with the presence of one transmembrane helical loop in one of the suggested models of the closed membrane channel. Selective labeling of the protein at single sites allows one to determine the limits and the orientation of the colicin helices. Distances have been measured accurately using REDOR magic angle spinning NMR spectroscopy on membrane proteins that are selectively labeled with 13C and 15N amino acids. This technique, therefore, provides valuable information on the global fold of proteins when associated with lipid bilayers. We gratefully acknowledge the support by Kathy Valentine and Stanley J. Opella as some of the spectra that will be presented have been acquired at the high-field magnet of the NIH resource for solid-state NMR of proteins.
E.Coli diacylglycerol kinase (DAGK) is a largely helical 122 residue protein which spans the cytoplasmic membrane three times (Sanders et.al., Biochem 35, 8610-8618) and which catalyzes the phosphorylation of a lipid, DAG. The structure of the fully functional enzyme in bilayers is being undertaken using several complementary techniques suited for membrane proteins, two of which are NMR-detected amide H-D exchange (AE) measurements and solid state NMR (OSNMR). Single-Cys mutants for each of 122 positions of DAGK were created after removing two native cysteine residues (Cysless-DAGK). After introducing single-Cys mutations, each mutant was specifically labeled using either 15N or 13C cysteine. Progress in AE and OSNMR studies of second transmembrane segment of DAGK is reported in this poster. Specifically, we describe the following. (i) A solvent exchange-trapping methodology employing 1H-15N HSQC to measure amide H-D exchange rates at cysteine sites within the second transmembrane segment (TM2) of DAGK in lipid bilayers; (ii) Oriented sample 15N, 13C, NMR spectra employing CP of single-cys mutants of DAGK (TM2) in magnetically-oriented bicelles to measure CSAs and dipolar couplings at each labeled site.
The investigation of the fluid exchange of methanol for liquid CO2 in nanoporous silica alcogels at pressures up to 10 MPa by NMR imaging and NMR spectroscopy requires a special designed high pressure probehead (1). This fluid exchange can be followed by time dependent NMR-imaging of the methanol in the alcogel. An intensitiy profile of NMR-images depends not only on the spin densitiy but also on the T1- and T2-values of the proton signal. The T1- and T2-corrected intensity profile yields the spin densitiy of methanol in the gel as a function of time during the fluid exchange. This time-dependent determination of the concentration-ratio of the mixture of methanol and liquid CO2 allows to determine the transport coefficient of methanol in the alcogel. This information is very important for the understanding of the transport in confined nanostructured systems as well as for the optimization of the preparation process for silica aerogels, i.e. the prediction of the duration of the fluid exchange (2). Since the T1- and T2-times and the self diffusion coefficients depend strongly on the concentration of methanol in liquid CO2, T1-, T2- and diffusion-maps have to be recorded additionally to the cross-section images of the gels during the fluid exchange. To get correct values of the spatial resolved spin density of methanol in the gel, some basic calibration measurements are necessary. T1- and T2-values and self diffusion coefficients of pure methanol as a function of the pressure applied (at fixed T = 15 C) determined via spectroscopic measurements will be presented . The same quantities are also studied as a function of the methanol / liquid CO2 ratio. Finally the maps of T1, T2 and the self diffusion coefficient of the methanol in the alcogels will be presented together with the cross-section images of gels with different porosity and different pH-value of the catalyst used upon preparation. In conclusion, all these values together are evaluated to determine the transport diffusion coefficient in situ. (1) A High-Pressure Probehead ..., 37th ENC, Orlando, 1997 (2) Self and Transport Diffusion of fluids in SiO2 Alcogels ..., to be published in J. Non-Cryst. Solids, 1998
We report here the results of 2H solid state NMR studies on water present in the interbilayer space of the phosphatidyl choline bilayers. Mechanically oriented DMPC bilayers were prepared by alligning the sample in between glass plates and the sample was fully hydrated with D2O to observe the behavior of water in between the bilayers. 2H NMR spectra were recorded both above and below the gel to liquid crystalline phase transition temperatures as well as sub-zero temperatures. Spectra above the gel to liquid crystalline phase transition temperature show a doublet apart from an isotropic line and the splitting of the doublet increases with increasing temperature. The doublet structure merges with the isotropic signal into a single line near the phase transition temperature. Spectra below the transition temperature show the single line splitting into a doublet once again and the splitting of this doublet increases with lowering temperature. Around 0 degrees C we can see at least two doublet structures developing. As the temperature is further lowered the individual lines broaden and join into a single line around -40 degrees C. There are indications for possible further splitting to appear in the spectrum around -70 degrees C, which we are in the process of verifying. Angular dependence and relaxation studies are further underway on this and other PC systems using the oriented sample technique to understand the conformation of the interbilayer water molecules, especially at low temperatures where the motional processes are expected to slow down considerably.
On this poster we describe the preparation of bicellar phospholipid solutions for a broad range of applications. The bicellar solutions vary from highly concentrated (20% phospholipid, 80% water) [1] to highly dilute (2% phospholipid, 98% water) [2], from neutral bicelles to bicelles with up to 25% anionic phospholipids, from discotic nematic bicelles to lanthanide doped, highly aligned smectic bilayers [3], from bicelles with low molar ratio (q=0.5 with q=long chain phospholipid / short chain phospholipid) for isotropic studies [4] to magnetically oriented bicelles with high q, suitable for measurements of residual dipolar and quadrupolar tensors for direct structure elucidation [5]. Seasoned by numerous trials and tribulations we can now present recipes for a great variety of tastes: Recipes for improving 'traditional' bicelles by adapting size and acidity of the bicelles to the pending task; recipes that include exotic ingredients such as differently flavored lanthanides; and recipes for highly dilute bicelles (varieties of the well-known Shirley Temple) for larger proteins. All are served with plenty of experimental data on an unusual and very versatile lyotropic liquid crystalline system which may be stimulating to all devotees of sophisticated drinks...errh,...complex fluids. References: [1] C.R. Sanders, and J.P. Schwonek, Biochemistry 31, 8898 (1992). [2] N. Tjandra and A. Bax, Science 278, 1111 (1997). [3] R.S. Prosser, J.S. Hwang and R.R.Vold, Biophysical Journal (in Press, 1998). [4] R.R. Vold, R.S.Prosser and A.J.Deese, J. Biomol. NMR 9, 329 (1997). [5] J.W. Emsley and J.C. Lindon, NMR Spectroscopy Using Liquid Crystal Solvents, Pergamon Press, Oxford 1975. Comment to the Organization: something or not enough happened with the software so: Yes The presentation represented in the presented abstract represents "new techniques". Thank you, Jo Struppe
Many important biological signaling processes occur in neural membranes that are rich in polyunsaturated acyl chains, such as 22:6n3 (DHA). Specific or nonspecific lipid-protein interactions may occur between membrane proteins and DHA acyl chains. We combined NMR and x-ray diffraction methods to study structural, dynamical, and elastic properties of the DHA chain in membrane lipids. With recent developments in high-resolution magic-angle-spinning (HR MAS) methods it has become possible to obtain structural and dynamic information from many positions on the DHA chain in bilayers without resorting to isotopic labeling. The 13C-1H dipolar interactions of all resolved 13C resonances in the DHA chain were determined by dipolar recoupling in HR-MAS NMR. Qualitative differences in motional correlation times along the DHA chain were evaluated from 13C spin-lattice relaxation times. X-ray diffraction was used to determine hydrocarbon chain length and area for lipids containing one or two DHA chains. Order parameters and average geometrical dimensions of the DHA chain are discussed in context with DHA conformations suggested by molecular modeling studies.