High-resolution proton and carbon-13 NMR of membranes: why sonicate?

We have obtained high-field (11.7-T) proton and carbon-13 Fourier transform (FT) nuclear magnetic resonance (NMR) spectra of egg lecithin and egg lecithin-cholesterol (1:1) multibilayers, using "magic-angle" sample spinning (MASS) techniques, and sonicated egg lecithin and egg lecithin-cholesterol (1:1) vesicles, using conventional FT NMR methods. Resolution of the proton and carbon-13 MASS NMR spectra of the pure egg lecithin samples is essentially identical with that of sonicated samples, but spectra of the unsonicated lipid, using MASS, can be obtained very much faster than with the more dilute, sonicated systems. With the 1:1 lecithin-cholesterol systems, proton MASS NMR spectra are virtually identical with conventional FT spectra of sonicated samples, while with 13C NMR, we demonstrate that most 13C nuclei in the cholesterol moiety can be monitored, even though these same nuclei are essentially invisible, i.e., are severely broadened, in the corresponding sonicated systems. In addition, 13C MASS NMR, spectra can again be recorded much faster than with sonicated samples, due to concentration effects. Taken together, these results strongly suggest there will seldom be need in the future to resort to ultrasonic disruption of lipid bilayer membranes in order to obtain high-resolution proton or carbon-13 NMR spectra.     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.     

Nuclear magnetic resonance studies of amino acids and proteins. Rotational correlation times of proteins by deuterium nuclear magnetic resonance spectroscopy

We show that measurement of the spin-lattice (T1) and spin-spin (T2) relaxation times (or line widths) of irrotationally bound 2H nuclei in macromolecules undergoing isotropic rotational motion outside of the extreme narrowing limit (i.e., for the case omega 02 tau R2 much greater than 1) permits determination of both the rotational correlation time (tau R) of the macromolecule and the electric quadrupole coupling constant (e2qQ/h) of the 2H label. The technique has the advantage over 13C nuclear magnetic resonance (NMR) that no assumptions about bond lengths (which appear to the sixth power in 13C relaxation studies) or relaxation mechanisms need to be made, since relaxation will always be quadrupolar, even for aromatic residues at high field. Asymmetry parameter (eta) uncertainties are shown to cause negligible effects on tau R determinations, and in any case it is shown that both e2qQ/h and eta may readily be determined in separate solid-state experiments. By way of example, we report 2H NMR results on aqueous lysozyme (EC 3.2.1.17) at 5.2 and 8.5 T (corresponding to 2H-resonance frequencies of 34 and 55 MHz). Interpretation of the results in terms of the isotropic rigid-rotor model yields e2qQ/h values of approximately equal to 170 or approximately equal to 190 kHz, respectively, for the imidazolium and free-base forms of [epsilon 1-2H] His-15 lysozyme in solution, in excellent agreement with e2qQ/h values of approximately 167 and approximately 190 kHz obtained for the free amino acids in the solid state. In principle, the method may in suitable cases permit comparison between the dynamic structures of proteins in solution and in the crystalline solid state.     

Spectroscopic studies of lipids and biological membranes: carbon-13 and proton magic-angle sample-spinning nuclear magnetic resonance study of glycolipid-water systems.

We have obtained 1H and 13C magic-angle sample-spinning (MAS) nuclear magnetic resonance (NMR) spectra of three glycosyldiacylglycerol-water (1:1, weight ratio) mesophases, at 11.7 T, as a function of temperature, in order to probe lipid headgroup, backbone, and acyl chain dynamics by using natural-abundance NMR probes. The systems investigated were monogalactosyldiacyldiglyceride [MGDG; primarily 1,2-di[(9Z,12Z,15Z)octadec-9,12,15-trienoyl++ +]-3-beta-D-galactopyranosyl- sn-glycerol]; digalactosyldiacyldiglyceride [DGDG; primarily 1,2-di[(9Z,12Z,15Z)octadec-9,12,15-trienoyl++ +]-3- (alpha-D-galactopyranosyl-1-6-beta-D-glactopyranosyl)-sn-glycerol] ; and sulfoquinovosyldiacyldiglyceride [SQDG; primarily 1-[(9Z,12Z,15Z)octadec-9,12,15-trienoyl]-2 -hexadecanoyl-3-(6-deoxyl-6- sulfono-alpha-D-glucopyranosyl)-sn-glycerol]. At approximately 22 degrees C, all three lipid-water systems give well-resoled 13C and 1H MAS NMR spectra, characteristic of fluid, liquid-crystalline mesophases. 13C spin-lattice relaxation times of the headgroup and glycerol backbone carbons of all three materials give, within experimental error, the same NT1 values (approximately 400 ms), implying similar high-frequency motions, independent of headgroup size and charge. Upon cooling, pronounced line broadenings are observed, due to an increase in slow motional behavior. For each lipid, the onset of line broadening is seen with the glycosyl headgroup, glycerol backbone, and the first two or three carbons of the acyl chains. By approximately -20 degrees, all headgroup carbon resonances are broadened beyond detection. Both galactose moieties in DGDG "freeze out" together, implying a rigid-body motion of the disaccharide unit. Upon further cooling, the bulk polymethylene chain resonances in all three systems (in both 13C and 1H MAS) broaden greatly, followed by the olefinic and allylic carbon resonances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the desulfurization genes from Rhodococcus sp. strain IGTS8.

Rhodococcus sp. strain IGTS8 possesses an enzymatic pathway that can remove covalently bound sulfur from dibenzothiophene (DBT) without breaking carbon-carbon bonds. The DNA sequence of a 4.0-kb BstBI-BsiWI fragment that carries the genes for this pathway was determined. Frameshift and deletion mutations established that three open reading frames were required for DBT desulfurization, and the genes were designated soxABC (for sulfur oxidation). Each sox gene was subcloned independently and expressed in Escherichia coli MZ1 under control of the inducible lambda pL promoter with a lambda cII ribosomal binding site. SoxC is an approximately 45-kDa protein that oxidizes DBT to DBT-5,5'-dioxide. SoxA is an approximately 50-kDa protein responsible for metabolizing DBT-5,5'-dioxide to an unidentified intermediate. SoxB is an approximately 40-kDa protein that, together with the SoxA protein, completes the desulfurization of DBT-5,5'-dioxide to 2-hydroxybiphenyl. Protein sequence comparisons revealed that the predicted SoxC protein is similar to members of the acyl coenzyme A dehydrogenase family but that the SoxA and SoxB proteins have no significant identities to other known proteins. The sox genes are plasmidborne and appear to be expressed as an operon in Rhodococcus sp. strain IGTS8 and in E. coli.     

Chemical shifts and three-dimensional protein structures

Summary During the past three years it has become possible to compute ab initio the ¹³C, ¹⁵N and ¹⁹F NMR chemical shifts of many sites in native proteins. Chemical shifts are beginning to become a useful supplement to more established methods of solution structure determination, and may find utility in solid-state analysis as well. From ¹³C NMR, information on , and torsions can be obtained, permitting both assignment verification, and structure refinement and prediction. For ¹⁵N, both torsional and hydrogen-bonding effects are important, while for ¹⁹F, chemical shifts are primarily indicators of the local charge field. Chemical shift calculations are still slow, but shielding hypersurfaces — the shift as a function of the dihedral angles that define the molecular conformation — are becoming accessible. Over the next few years, theoretical and computer hardware improvements will enable more routine use of chemical shifts in structural studies, including the study of metal-ligand interactions, the analysis of drug and substrate binding and catalysis, the study of folding/unfolding pathways, as well as the characterization of conformational substates. Rather than simply being a necessary prerequisite for multidimensional NMR, chemical shifts and chemical shift non-equivalence due to folding are now beginning to be useful for structural characterization.     

Protein-lipid interactions. A nuclear magnetic resonance study of sarcoplasmic reticulum Ca2,Mg2+-ATPase, lipophilin, and proteolipid apoprotein-lecithin systems and a comparison with the effects of cholesterol.

Deuterium Fourier transform nuclear magnetic resonance (NMR) spectra at 34 MHz (corresponding to a magnetic field strength of 5.2 T) have been obtained of a variety of protein-lipid systems containing specifically deuterated phospholipids. The following systems were investigated as a function of temperature: sarcoplasmic reticulum ATPase (ATP phosphohydrolase, EC 3.6.1.3) complexed with 1-myristoyl-2-(14,14,14-trideuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d3) or 1,2-bis(16,16,16-trideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-k6); human brain lipophilin complexed with DPPC-d6 or 1,2-bis(6,6-dideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-6,6-d4); beef brain myelin proteolipid apoprotein (PLA) reconstituted with DMPC labeled as CD2 (or CD3) at one or more of positions 3, 4, 6, 8, 10, 12, or 14 of the sn-2 chain. For purposes of comparison, spectra were also obtained for bilayers containing cholesterol (CHOL). The results show that proteins either disorder or have little effect on hydrocarbon chain order in membranes above the gel to liquid-crystal phase transition temperature (Tc) of the pure lipids. Cholesterol, however, causes a very large ordering of the hydrocarbon chains above Tc, but both cholesterol and protein prevent chain crystallization (by effectively disordering chain packing) immediately below Tc. No evidence for any ordered "boundary lipid" in association with protein was found above Tc, perhaps due to the rough nature of protein surfaces. Above Tc, exchange between free bilayer and protein associated lipid is fast on the time scale of the deuterium NMR experiment (greater than or similar to 10(3) s-1). We have also obtained proton-decoupled phosphorus-31 nuclear magnetic resonance spectra at 60.7 MHz (corresponding to a magnetic field strength of 3.5 T) of DMPC, DMPC-AT-Pase, and DMPC-CHOL complexes. The results indicate that ATPase and CHOL CAUSE SMALL DECREASES IN 31P chemical shielding anisotropies but that in addition ATPase causes a four- to fivefold increase in 31P spin-lattice and Carr-Purcell spin-spin relaxation rates, suggesting the possibility of polar group protein-lipid interaction leading to increased correlation times in the region of the lipid phosphate head group.