Different distribution of fluorinated anesthetics and nonanesthetics in model membrane: a 19F NMR study.

Despite their structural resemblance, a pair of cyclic halogenated compounds, 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1,2-dichlorohexafluorocyclobutane (F6), exhibit completely different anesthetic properties. Whereas the former is a potent general anesthetic, the latter produces no anesthesia. Two linear compounds, isoflurane and 2,3-dichlorooctofluorobutane (F8), although not a structural pair, also show the same anesthetic discrepancy. Using 19F nuclear magnetic spectroscopy, we investigated the time-averaged submolecular distribution of these compounds in a vesicle suspension of phosphatidylcholine lipids. A two-site exchange model was used to interpret the observed changes in resonance frequencies as a function of the solubilization of these compounds in membrane and in water. At clinically relevant concentrations, the anesthetics F3 and isoflurane distributed preferentially to regions of the membrane that permit easy contact with water. The frequency changes of these two anesthetics can be well characterized by the two-site exchange model. In contrast, the nonanesthetics F6 and F8 solubilized deeply into the lipid core, and their frequency change significantly deviated from the prediction of the model. It is concluded that although anesthetics and nonanesthetics may show similar hydrophobicity in bulk solvents such as olive oil, their distributions in various regions in biomembranes, and hence their effective concentrations at different submolecular sites, may differ significantly.     

Difluorophosphate as a 19F NMR probe of erythrocyte membrane potential

Erythrocyte membrane potential can be estimated by measuring the transmembrane concentration (activity) distribution of a membrane-permeable ion. We present here the study of difluorophosphate (DFP) as a ¹⁹F NMR probe of membrane potential. This bicarbonate and phosphate analogue has a pK_a of 3.7±0.2 (SD, n = 4) and therefore exists almost entirely as a monovalent anion at physiological pH. When it is incorporated into red cell suspensions, it gives two well resolved resonances that arise from the intra- and extracellular populations; the intracellular resonance is shifted 130 Hz to higher frequency from that of the extracellular resonance. Hence the transmembrane distribution of DFP is readily assessed from a single ¹⁹F NMR spectrum and the membrane potential can be calculated using the Nernst equation. The membrane potential was independent of, DFP concentration in the range 4 to 59 mM, and haematocrit of the cell suspensions of 31.0 to 61.4%. The membrane potential determined by using DFP was 0.94±0.26 of that estimated from the transmembrane pH difference. The distribution ratios of intracellular/extracellular DFP were similar to those of the membrane potential probes, hypophosphite and trifluoroacetate. DFP was found to be transported across the membranes predominantly via the electrically-silent pathway mediated by capnophorin. Using magnetization transfer techniques, the membrane influx permeability-coefficient of cells suspended in physiological medium was determined to be 7.2±2.5 × 10^–6 cm s^–1 (SD, n=4). Offprint requests to: P. W Kuchel     

The measurement of immersion depth and topology of membrane proteins by solution state NMR

An important component of the study of membrane proteins involves the determination of details associated with protein topology — for example, the location of transmembrane residues, specifics of immersion depth, orientation of the protein in the membrane, and extent of solvent exposure for each residue. Solution state NMR is well suited to the determination of immersion depth with the use of paramagnetic additives designed to give rise to depth-specific relaxation effects or chemical shift perturbations. Such additives include spin labels designed to be “anchored” within a given region of the membrane or small freely diffusing paramagnetic species, whose partitioning properties across the water membrane interface create a gradient of paramagnetic effects which correlate with depth. This review highlights the use of oxygen and other small paramagnetic additives in studies of immersion depth and topology of membrane proteins in lipid bilayers and micelles.     

The measurement of immersion depth and topology of membrane proteins by solution state NMR

An important component of the study of membrane proteins involves the determination of details associated with protein topology - for example, the location of transmembrane residues, specifics of immersion depth, orientation of the protein in the membrane, and extent of solvent exposure for each residue. Solution state NMR is well suited to the determination of immersion depth with the use of paramagnetic additives designed to give rise to depth-specific relaxation effects or chemical shift perturbations. Such additives include spin labels designed to be "anchored" within a given region of the membrane or small freely diffusing paramagnetic species, whose partitioning properties across the water membrane interface create a gradient of paramagnetic effects which correlate with depth. This review highlights the use of oxygen and other small paramagnetic additives in studies of immersion depth and topology of membrane proteins in lipid bilayers and micelles.     

Determination of membrane potential and cell volume by 19F NMR using trifluoroacetate and trifluoroacetamide probes

The distribution of ionic species between intra- and extracellular compartments forms one basis for the determination of cell membrane potential. It is shown that fluorine-19 NMR studies of erythrocytes in the presence of trifluoroacetate, a stable, relatively nontoxic anion with pK = -0.3, provide a sensitive probe of membrane potential. Since such measurements are based on ion concentrations, the parallel use of the neutral analogue trifluoroacetamide to provide information on intra/extracellular volume ratios was also explored. In both cases, separate 19F resonances corresponding to intra- and extracellular ions were observed, with the intracellular resonance shifted downfield by approximately 0.2 ppm and the intracellular peak typically somewhat broader than the extracellular resonance. Studies with the band 3 anion-exchange inhibitor 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) indicate that both transmembrane diffusion and flux involving the band 3 anion exchanger contribute to the observed transport of the trifluoroacetate anion. Intra/extracellular volume ratios determined on the basis of trifluoroacetamide intensity ratios were in good agreement with determinations based on measured hematocrits. On the basis of the high sensitivity of 19F NMR and the capability of monitoring volume changes simultaneously, the time resolution for these measurements can approach the lifetime of intracellular trifluoroacetate ions and hence be limited by the trifluoroacetate flux rate.     

Interaction of trifluoperazine with Tetrahymena calmodulin. A 19F NMR study

We have used 19F NMR to study interactions of trifluoperazine (TFP), a potent calmodulin (CaM) antagonist, with Tetrahymena calmodulin (Tet. CaM). Changes in chemical shift and bandwidth of TFP caused by adding Tet. CaM in the presence of excess Ca2+ were much smaller than those by adding porcine CaM. The spectral features of the TFP-Tet. CaM solution in the presence of excess Ca2+ were quite similar to those of the TFP-porcine CaM solution in the absence of Ca2+. The exchange rate of TFP from Tet. CaM was estimated to be nearly 20 s-1. The TFP-Tet. CaM solution in the absence of Ca2+ showed a pronounced pH dependence of the 19F NMR chemical shift, whereas the solution in the presence of excess Ca2+ showed a smaller pH dependence. Thus, it was suggested that TFP is located near a hydrophilic region of the Tet. CaM molecule in the absence of Ca2+, while TFP is located near a hydrophobic region of the Tet. CaM in the presence of excess Ca2+.     

19F NMR investigation of F(1)-ATPase of Escherichia coli using fluorotryptophan labeling

Growth of Escherichia coli in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, has permitted the production of proton-dislocating ATPase that is specifically labeled with 5-fluorotryptophan. Five sets of (19)F resonances could be assigned to each tryptophan residue by lauryldimethylamine oxide and carboxypeptidase treatment. On labeling with 4-chloro-7-nitro-benzofurazan, the label attached to b155Lys, which is known to be in the catalytic site, which caused one of the residues, b108Trp, to become nonequivalent. (19)F NMR spectroscopic investigation of internally fluorotryptophan-labeled F(1)-ATPase will provide valuable information about the asymmetric nature of F(1)-ATPase and the conformational changes induced by ligand binding.     

Advances in the study of GPCRs by 19F NMR

Crystallography and cryo-electron microscopy have advanced atomic resolution perspectives of inactive and active states of G protein-coupled receptors (GPCRs), alone and in complex with G proteins or arrestin. ¹⁹F NMR can play a role in ascertaining activation mechanisms and understanding the complete energy landscape associated with signal transduction. Fluorinated reporters are introduced biosynthetically via fluorinated amino acid analogs or chemically, via thiol-specific fluorinated reporters. The chemical shift sensitivity of these reporters makes it possible to discern details of conformational ensembles. In addition to spectroscopic details, paramagnetic species can be incorporated through orthogonal techniques to obtain distance information on fluorinated reporters, while T₂-and T₁-based relaxation experiments provide details on exchange kinetics in addition to fluctuations within a given state.     

Reactivity of Al(III) with membrane phospholipids: a NMR approach

The complexes Al(acac)₃ (1) (acac = 2,4-pentanedionate) and Al(malt)₃ (malt = 3-hydroxy-2-methyl-4-pyronate) (2) react with dl--dipalmitoylphosphatidylcholine (DPPC) under a 1:1 molar ratio in CDCl₃ at 37 °C, as shown by the substantial release of ligands (20–50%) from the metal coordination sphere (¹H-NMR), by evident changes in the ¹H-NMR spectrum of DPPC in the reaction mixture and by the appearance of a ³¹P-NMR signal due to metal-coordinated DPPC. ³¹P-NMR spectra reveal that both 1 and 2 also react with DPPC in water, in the presence of 1% Triton X-100 and Tris buffer. Under these conditions, 1 and 2 do not react with ghosts from human erythrocytes. On the contrary, the far less hydrolytically stable complex Al(lact)₃ (lact = lactate) appears to be reactive under identical conditions, as shown by ³¹P-NMR spectra.     

In situ 19F NMR studies of an E. coli membrane protein

In this report, (19)F spin incorporation in a specific site of a specific membrane protein in E. coli was accomplished via trifluoromethyl-phenylalanine ((19) F-tfmF). Site-specific (19)F chemical shifts and longitudinal relaxation times of diacylglycerol kinase (DAGK), an E. coli membrane protein, were measured in its native membrane using in situ magic angle spinning (MAS) solid state nuclear magnetic resonance (NMR). Comparing with solution NMR data of the purified DAGK in detergent micelles, the in situ MAS-NMR data illustrated that (19)F chemical shift values of residues at different membrane protein locations were influenced by interactions between membrane proteins and their surrounding lipid or lipid mimic environments, while (19)F side chain longitudinal relaxation values were probably affected by different interactions of DAGK with planar lipid bilayer versus globular detergent micelles.     

Investigation of the Ca2+-dependent interaction of trifluoperazine with S100a: A19F NMR and circular dichroism study

S100a is a heterodimeric, acidic calcium-binding protein that interacts with calmodulin antagonists in a Ca²⁺-dependent manner. In order to study the behavior of the hydrophobic domain on S100a when bound to Ca²⁺, its interaction with trifluoperazine (TFP) was investigated using¹⁶F nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The dissociation constant (K _d) values of TFP, as estimated from the chemical shifts of¹⁹F NMR, were 191 and 29 μm in the absence and presence of Ca²⁺, respectively, and were similar to those previously reported for S100b. However, the TFP linewidth in the presence of Ca²⁺-bound S100a was 65 Hz greater than in the presence of Ca²⁺-bound S100b. This suggests a slower TFP exchange rate for S100a than for S100b. Thus, the TFP linewidths observed for each isoform may reflect differences in structural and modulatory properties of the Ca²⁺-dependent hydrophobic domains on S100a and S100b. Additionally, the presence of magnesium had no effect on the observed Ca²⁺-induced TFP spectral changes in S100a solutions. Circular dichroism studies indicate that Ca²⁺ induces a small transition from α-helix to random coil in S100a; in contrast, the opposite transition is reported for calmodulin (Hennesseyet al., 1987). However, TFP did not significantly alter the secondary structure of Ca²⁺-bound S100a; this observation is similar to the effect of TFP on Ca²⁺-bound calmodulin and troponin C (Shimizu and Hatano, 1984; Gariépy and Hodges, 1983). It is, therefore, proposed that TFP binds to a hydrophobic domain on S100a in a fashion similar to other calcium-modulated proteins.     

19F NMR study on the biological Baeyer-Villiger oxidation of acetophenones

The biological Baeyer–Villiger oxidation of acetophenones was studied by ¹⁹F nuclear magnetic resonance (NMR). The ¹⁹F NMR method was used to characterise the time-dependent conversion of various fluorinated acetophenones in either whole cells of Pseudomonas fluorescens ACB or in incubations with purified 4′-hydroxyacetophenone monooxygenase (HAPMO). Whole cells of P. fluorescens ACB converted 4′-fluoroacetophenone to 4-fluorophenol and 4′-fluoro-2′-hydroxyacetophenone to 4-fluorocatechol without the accumulation of 4′-fluorophenyl acetates. In contrast to 4-fluorophenol, 4-fluorocatechol was further degraded as evidenced by the formation of stoichiometric amounts of fluoride anion. Purified HAPMO catalysed the strictly NADPH-dependent conversion of fluorinated acetophenones to fluorophenyl acetates. Incubations with HAPMO at pH 6 and 8 showed that the enzymatic Baeyer–Villiger oxidation occurred faster at pH 8 but that the phenyl acetates produced were better stabilised at pH 6. Quantum mechanical characteristics explained why 4′-fluoro-2′-hydroxyphenyl acetate was more sensitive to base-catalysed hydrolysis than 4′-fluorophenyl acetate. All together, ¹⁹F NMR proved to be a valid method to evaluate the biological conversion of ring-substituted acetophenones to the corresponding phenyl acetates, which can serve as valuable synthons for further production of industrially relevant chemicals. Journal of Industrial Microbiology & Biotechnology (2001) 26, 35–42. Received 20 April 2000/ Accepted in revised form 16 September 2000     

(19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution

The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein.     

Conformational changes in the human estrogen receptor observed by (19)F NMR

The (19)F NMR spectra of the 5F-Trp labeled glutathione-S-transferase fusion protein with residues 282-595 of the human estrogen receptor show that there is a distinct conformational change in the protein when estradiol is added to the unliganded protein. Our studies show the empty receptor to have more conformational flexibility than the liganded form. This study shows the applicability of (19)F NMR to study conformational change in large protein systems.     

19F NMR studies of the D-galactose chemosensory receptor. 2. Ca(II) binding yields a local structural change.

The Escherichia coli D-galactose and D-glucose receptor possesses a Ca(II)-binding site closely related in structure and metal-binding characteristics to the eukaryotic EF-hand sites. Only the structure of the Ca(II)-occupied site is known. To investigate the structural change triggered by Ca(II) and Sr(II) binding, we have used 19F NMR to probe five 5-fluorotryptophan (5F-Trp) and seven 3-fluorophenylalanine (3F-Phe) positions in the structure, extending the approach described in the preceding article. Of particular interest were two 5F-Trp residues near the N terminus of the Ca(II) site at positions 127 and 133. Substitution of the larger Sr(II) for Ca(II) triggered 19F NMR frequency shifts of the 5F-Trp127 and -133 resonances, indicating a detectable structural change in the Ca(II) site. In contrast, the three 5F-Trp resonances from distant regions of the structure exhibited no detectable frequency shifts. When the metal was removed from the Ca(II) site, the 5F-Trp127 and -133 frequencies shifted to a new value similar to that observed for free 5F-Trp in aqueous solvent, and this new frequency was a function of the H2O to D2O ratio, indicating that the residues had become solvent exposed. Metal removal yielded small or undetectable frequency shifts for the three distant 5F-Trp resonances and for four of the five resolved 3F-Phe resonances. The allosteric coupling of the metal and sugar binding sites was observed to be slight: depletion of metal ions was observed to reduce the D-galactose affinity of the receptor by 2-fold. Together the results indicate that the structural changes in the Ca(II) site are primarily localized in the region of the site. Removal of the metal ion from the site exposes the nearby 5F-Trp127 and -133 residues to the solvent, suggesting that the empty site has a more open structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

19F NMR study of the interactions of fluoride with superoxide dismutase and hemoglobin in erythrocytes

F- added to an erythrocyte suspension shows two separated resonances arisen from intra and extracellular compartments. Cu, Zn superoxide dismutase dominates the longitudinal relaxation rate of the intracellular F- resonance, while diamagnetic interactions with hemoglobin contribute mainly to the transversal relaxation rate.     

Solubility of cyclomaltooligosaccharides (cyclodextrins) in H2O and D2O: a comparative study

Cyclomaltooligosaccharides (cyclodextrins, CDs) are cyclic oligomers having six, seven, or eight units of alpha-D-glucose, named as cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD) and cyclomaltooctaose (gamma-CD), respectively. The molecule of CD has a cavity in which the interior is hydrophobic relative to its outer surface. The solubility of cyclodextrins in water is unusual, as an irregular trend is observed in the series of the cyclic oligomers of glucose. beta-CD is at least nine times less soluble than the others CDs. This intriguing behavior has been investigated, and some interesting explanations in terms of the effect caused by CD on the water lattice structure have been proposed. In this work a comparative study on the solubility of alpha, beta, and gamma-cyclodextrins was carried out in H2O and D2O and reveals a much lower solubility of the three CDs in D2O. The solid-phase structure of the CDs in equilibrium with the solution is quite similar with both solvents. The results are discussed in terms of the CD molecular structure and the differences in the hydrogen bonds formed between H2O and D2O.     

19F NMR study of the myosin and tropomyosin binding sites on actin

Actin was labeled with pentafluorophenyl isothiocyanate at Lys-61. The label was sufficiently small not to affect the rate or extent of actin polymerization unlike the much larger fluorescein 5-isothiocyanate which completely inhibits actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. Furthermore, the label resonances in the 376.3-MHz 19F NMR spectrum were unaffected by actin polymerization. However, the binding of the relaxing protein tropomyosin resulted in the fluorinated Lys-61 resonances broadening out beyond detection due to a substantial increase in the effective correlation time of the label. Similarly, the binding of myosin subfragment 1 to F-actin resulted in the dramatic broadening of the labeled Lys-61 resonances. Thus, Lys-61 on actin appears to be closely associated with the binding sites for both tropomyosin and myosin, suggesting that both these proteins can compete for the same site on actin. The other region of actin known to be involved in myosin binding, Cys-10, was found to be more remote from the actin-actin interfaces than Lys-61. Labels on Cys-10 exhibited substantially greater mobility than fluorescein 5-isothiocyanate attached to Lys-61 which appeared to be held down on the surface of the actin monomer. This may sterically hinder the actin-actin interaction about 1 nm from the tropomyosin/myosin binding site.     

19F NMR study on the biodegradation of fluorophenols by various Rhodococcus species

Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.