Interaction of tryptophan with lecithin liposomes: NMR and turbidity studies

A study on the interactions between tryptophan (Trp) and dipalmitoylphosphatidyl choline (DPPC) liposomes conducted with the NMR technique and taking turbidity measurements is reported. Trp is shown to be incorporated into the bilayer only when interaction occurs above gel-liquid transition. Disappearance of turbidity changes at the phase transition temperatures are shown to occur with Trp incorporation. 1H and 13C NMR relaxation times T1 of DPPC are seen to be reduced. Acyl chain signal intensity is shown to decrease and the corresponding line-width to increase as a function of Trp concentration. DPPC 31P [1H] Nuclear Overhauser Effect (NOE) is depressed by the presence of Trp above gel-liquid transition temperature whereas NOE remains high below phase transition temperature when Trp is present in the bilayer. Effects are shown to be the same in both H2O and in 2H2O. A membrane modification that may account for the previously observed inhibition of polysaccharide induced cell aggregation is hypothesized.     

Interaction of local anesthetics with small phospholipid vesicles investigated by proton NMR spectroscopy

The effects of the local anesthetics tetracaine, procaine (both charged at pH 6), and benzocaine (uncharged) on phospholipid liposomes have been investigated by 500 MHz ¹H NMR Spectroscopy. All the drugs reverse the Pr³⁺ induced shifts of phospholipid resonances in the same sequence as they are shifted by addition of Pr³⁺: choline POCH₂- > choline-CH₂N > choline-N(CH₃)₃ > glycerol > glycerol > acyl C₂ > acyl C₃. The drug effects result from incorporation of positive charges (tetracaine and procaine) and from the induction of a conformational change of the phospholipid head group via an action on the lipid glycerol backbone (benzocaine). From titration experiments with tetracaine on liposomes containing Pr³⁺ inside and outside is derived that the drug passes the bilayer by transverse diffusion. Tetracaine partitions outside/inside at a ratio of 21. Changes in linewidths of the drug resonances when incorporated into the liposomes allow the conclusion that the tetracaine molecule is located in an elongated way between the lipid acyl chains with its nitrogen group near the glycerol backbone. Benzocaine, showing strong effects on the line shapes of the protons on C₂ and C₃ of the lipid acyl chains is also located near the glycerol backbone, the region with the strongest hydrophobic forces.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 30), Cardiology.     

Interaction of the Saccharomyces cerevisiae alpha-factor with phospholipid vesicles as revealed by proton and phosphorus NMR

Proton and phosphorus-31 nuclear magnetic resonance (1H and 31P NMR) studies of the interaction between a tridecapeptide pheromone, the alpha-factor of Saccharomyces cerevisiae, and sonicated lipid vesicles are reported. 31P NMR studies demonstrate that there is interaction of the peptide with the phosphorus headgroups, and quasielastic light scattering (QLS) studies indicate that lipid vesicles increase in size upon addition of peptide. Previous solution (aqueous and DMSO) studies from this laboratory indicate that alpha-factor is highly flexible with only one long-lived identifiable structural feature, a type II beta-turn spanning the central portion of the peptide. Two-dimensional (2D) 1H nuclear Overhauser effect spectroscopy (NOESY) studies demonstrate a marked ordering of the peptide upon interaction with lipid, suggesting a compact N-terminus, in addition to a stabilized beta-turn. In contrast to our results in both solution and lipid environment, Wakamatsu et al. [Wakamatsu, K., Okada, A., Suzuki, M., Higashijima, T., Masui, Y., Sakakibara, S., & Miyazawa, T. (1986) Eur. J. Biochem. 154, 607-615] proposed a lipid environment conformation, on the basis of one-dimensional transferred NOE studies in D2O, which does not include the beta-turn.     

Comparison of the conformation of an oligonucleotide containing a central G-T base pair with the non-mismatch sequence by proton NMR.

We have recorded NOESY spectra of two non-selfcomplementary undecanucleotide duplexes. From the observed NOEs we do not detect any significant distortion of the helix when a G-C pair is replaced by a G-T pair and the normal interresidue connectivities can be followed through the mismatch site. We conclude that the 2D spectra of the non-exchangeable protons do not allow differentiation between a wobble or rare tautomer form for the mismatch. NOE measurements in H2O, however, clearly show that the mismatch adopts a wobble structure and give information on the hydration in the minor groove for the G-T base pair which is embedded between two A-T base pairs in the sequence.     

Nature of lysozyme-water interactions by proton NMR.

Proton spin-lattice relaxation measurements were performed in 10 mM lysozyme solution as a function of temperature and degree of substitution of solvent H2O with D2O. The results show that in the temperature range from 274 to 323 K, the intermolecular lysozyme proton water proton coupling contributes appreciably to the observed water proton relaxation rate. In this system exchange between water protons and labile protein protons does not dominate the behaviour with temperature of the water-lysozyme intermolecular contribution to the spin-lattice relaxation.     

Recognition of D-homoannulation by proton and carbon NMR spectra

One-and two dimensional proton and carbon NMR spectra of the D-homoannulated rearrangement product of triamcinolone (9 alpha-fluoro-11 beta,16 alpha,17 alpha, 21-tetrahydroxy-pregna-1,4-diene-3,20-dione) establish its structure as that of 9 alpha-fluoro-11 beta,16 alpha,17 alpha-trihydroxy-17 beta-hydroxy-methyl-D-homoandrosta-1,4-diene-3,17 alpha-dione. These methods accord ready recognition of D-homoannulation of C21-17-hydroxy-20-ketosteroids.     

Characterization of the liquid-ordered state by proton MAS NMR

We investigated if magic angle spinning (MAS) 1H NMR can be used as a tool for detection of liquid-ordered domains (rafts) in membranes. In experiments with the lipids SOPC, DOPC, DPPC, and cholesterol we demonstrated that 1H MAS NMR spectra of liquid-ordered domains (lo) are distinctly different from liquid-disordered (ld) and solid-ordered (so) membrane regions. At a MAS frequency of 10 kHz the methylene proton resonance of hydrocarbon chains in the ld phase has a linewidth of 50 Hz. The corresponding linewidth is 1 kHz for the lo phase and several kHz for the so phase. According to results of 1H NMR dipolar echo spectroscopy, the broadening of MAS resonances in the lo phase results from an increase in effective strength of intramolecular proton dipolar interactions between adjacent methylene groups, most likely because of a lower probability of gauche/trans isomerization in lo. In spectra recorded as a function of temperature, the onset of lo domain (raft) formation is seen as a sudden onset of line broadening. Formation of small domains yielded homogenously broadened resonance lines, whereas large lo domains (diameter >0.3 microm) in an ld environment resulted in superposition of the narrow resonances of the ld phase and the much broader resonances of lo. 1H MAS NMR may be applied to detection of rafts in cell membranes.     

Oxidation of tryptophan to oxindolylalanine by dimethyl sulfoxide-hydrochloric acid. Selective modification of tryptophan containing peptides

Tryptophan is readily oxidized to oxindolylalanine (2-hydroxytryptophan) in good yield on treatment in acetic acid solution with a mixture of dimethyl sulfoxide (DMSO) and concentrated aqueous HCl at room temperature. Other sulfoxides can be used in combination with HCl; for example, methionine sulfoxide reacts with an equimolar amount of tryptophan to give high yields of methionine and oxindolylalanine. Methionine and cysteine are quantitatively oxidized by DMSO/HCl to methionine sulfoxide and cystine, respectively. The tryptophan containing peptides LRF (luteinizing hormone-releasing factor), somatostatin, valine-gramicidin A and ACTH 1-24 were each treated with the DMSO/HCl reagent in acetic acid solution and the corresponding oxindolylalanine-derivatives isolated in over 90% yield after chromatography. The identity and purity of the derivatives were established on the basis of ultraviolet spectral characteristics and quantitative amino acid analysis of the oxindolylalanine content of acid hydrolyzates of the oxidized peptides with 3N-p-toluenesulfonic acid at 110 degrees for 24 h. The results indicate that modification of tryptophan peptides with DMSO/HCl provides a useful procedure, which seems superior to previously used reagents. In addition, the method could be well applied to other indoles of biological and pharmacological interest.     

Interaction of charged amphiphilic drugs with phosphatidylcholine vesicles studied by NMR

Small unilamellar vesicles from egg phosphatidylcholine in NaCl solutions were exposed to some amphiphilic pharmaca. The aromatic drugs (chlorpromazine, dibucaine, tetracaine, imipramine and propranolol) were in their cationic form of the amines. By 1H- (100 and 400 MHz) and 31P- (40.5 and 161.7 MHz) NMR the membrane signals were observed. In particular, the N-methyl choline proton signals were followed upon drug addition. The intrinsic chemical shift difference (0.02 ppm) between the inner (upfield) and outer choline signals was influenced by the drug concentration. Packing properties of the lipid head groups and ring current shift probably contributed. At very high drug concentration, the vesicles are destroyed. A transformation into a micellar state with a high sample viscosity took place in a narrow concentration range of drug. The anion effects of Cl- were studied from the 35Cl-NMR linewidth at 9.8 and 39.1 MHz. A continuous increase in the signal linewidth followed upon drug addition to the vesicles. Only chlorpromazine produced a broadening in the absence of vesicles (NaCl blank). The linewidth reflected a critical micelle concentration of this drug around 7 mM in 0.1 M NaCl. The 35Cl-NMR experiments demonstrated the existence of an anionic counterion effect. This phenomenon should be accounted for when quantitatively analysing drug-membrane interactions in electrostatic terms.     

Interaction of statins with phospholipid bilayers studied by solid-state NMR spectroscopy

Statins are drugs that specifically inhibit the enzyme HMG-CoA reductase and thereby reduce the concentration of low-density lipoprotein cholesterol, which represents a well-established risk factor for the development of atherosclerosis. The results of several clinical trials have shown that there are important intermolecular differences responsible for the broader pharmacologic actions of statins, even beyond HMG-CoA reductase inhibition. According to one hypothesis, the biological effects exerted by these compounds depend on their localization in the cellular membrane. The aim of the current work was to study the interactions of different statins with phospholipid membranes and to investigate their influence on the membrane structure and dynamics using various solid-state NMR techniques. Using ¹H NOESY MAS NMR, it was shown that atorvastatin, cerivastatin, fluvastatin, rosuvastatin, and some percentage of pravastatin intercalate the lipid-water interface of POPC membranes to different degrees. Based on cross-relaxation rates, the different average distribution of the individual statins in the bilayer was determined quantitatively. Investigation of the influence of the investigated statins on membrane structure revealed that lovastatin had the least effect on lipid packing and chain order, pravastatin significantly lowered lipid chain order, while the other statins slightly decreased lipid chain order parameters mostly in the middle segments of the phospholipid chains.     

Phase-resolved spectral measurements with several two tryptophan containing proteins

We have used frequency domain fluorescence techniques to resolve the component emission spectra for several two tryptophan containing proteins (e.g., horse liver alcohol dehydrogenase, sperm whale apomyoglobin, yeast 3-phosphoglycerate kinase, apoazurin from Alcaligenes denitricans). We have first performed multifrequency phase/modulation measurements and have found the fluorescence of each of these proteins to be described by a double exponential. Then, using phase-sensitive detection and the algorithm of Gratton and Jameson [Gratton, E., & Jameson, D. M. (1985) Anal. Chem. 57, 1694-1697], we have determined the emission spectrum associated with each decay time for these proteins. We have compared these phase-resolved spectra with the fractional contributions of the component fluorophores determined by selective solute quenching experiments. Reasonably good agreement is seen in most cases, which argues that the individual Trp residues emit independently. In the case of apoazurin, however, a negative amplitude is seen for the phase-resolved spectrum of the short-lifetime component. This pattern is consistent with the occurrence of energy transfer from the internal Trp residue to the surface Trp of this protein. We also present multifrequency lifetime measurements, phase-resolved spectra, and solute quenching data for a few protein-ligand complexes, to illustrate the utility of this approach for the study of changes in the fluorescence of proteins.     

Helical stacking in DNA three-way junctions containing two unpaired pyrimidines: proton NMR studies.

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.     

Carbon‐11 radiolabeling of an oligopeptide containing tryptophan hydrochloride via a Pictet‐Spengler reaction using carbon‐11 formaldehyde

A procedure for the synthesis of a¹¹C‐labeled oligopeptide containing [1‐¹¹C]1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ([1‐¹¹C]Tpi) from the corresponding Trp?HCl‐containing peptides has been developed involving a Pictet‐Spengler reaction with [¹¹C]formaldehyde. The synthesis of [1‐¹¹C]Tpi from Trp and [¹¹C]formaldehyde was examined as a model reaction with the aim of developing a facile and effective method for the labeling of peptides with carbon‐11. The Pictet‐Spengler reaction of Trp and [¹¹C]formaldehyde in acidic media (TsOH or HCl) afforded the desired [1‐¹¹C]Tpi in a moderate radiochemical yield. Herein, the application of a Pictet‐Spengler reaction to an aqueous solution of Trp?HCl gave the desired product with a radiochemical yield of 45.2%. The RGD peptide cyclo[Arg‐Gly‐Asp‐D‐Tyr‐Lys] was then selected as a substrate for the labeling reaction with [¹¹C]formaldehyde. The radiolabeling of a Trp?HCl‐containing RGD peptide using the Pictet‐Spengler reaction was successful. Furthermore, the remote‐controlled synthesis of a [1‐¹¹C]Tpi‐containing RGD peptide was attempted by using an automatic production system to generate [¹¹C]CH₃I. The radiochemical yield of the [1‐¹¹C]Tpi‐containing RGD at the end of synthesis (EOS) was 5.9 ± 1.9% (n = 4), for a total synthesis time of about 35 min. The specific activity was 85.7 ± 9.4 GBq/µmol at the EOS. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Amide proton exchange rates of a bound pepsin inhibitor determined by isotope-edited proton NMR experiments

From a series of isotope-edited proton NMR spectra, amide proton exchange rates were measured at 20 degrees C, 30 degrees C, and 40 degrees C for a tightly bound 15N-labeled tripeptide inhibitor of porcine pepsin (IC50 = 1.7 X 10(-) M). Markedly different NH exchange rates were observed for the three amide protons of the bound inhibitor. The P1 NH exchanged much more slowly than the P2 NH and P3 NH. These results are discussed in terms of the relative solvent accessibility in the active site and the role of the NH protons of the inhibitor for hydrogen bonding to the enzyme. In this study a useful approach is demonstrated for obtaining NH exchange rates on ligands bound to biomacromolecules, the knowledge of which could be of potential utility in the design of therapeutically useful nonpeptide enzyme inhibitors from peptide leads.