Molecular basis for the anti-sickling activity of aromatic amino acids and related compounds: a proton nuclear magnetic resonance investigation

High-resolution proton nuclear magnetic resonance spectroscopy and relaxation techniques have been used to investigate the interactions of sickle cell hemoglobin (Hb S) and human normal adult hemoglobin (Hb A) with p-bromobenzyl alcohol, L-phenylalanine, L-tryptophan, and L-valine. With the exception of valine, all these compounds inhibit the polymerization of deoxy-Hb S [Noguchi, C. T., & Schechter, A. N. (1978) Biochemistry 17, 5455)). Using transferred nuclear Overhauser effects among the proton resonances of the compound of interest and the corresponding longitudinal relaxation rates (T1(-1], we have shown that the binding of each of the compounds investigated to deoxy-Hb S is comparable to that to deoxy-Hb A. Intermolecular transferred nuclear Overhauser effects have been observed between proton resonances of the anti-sickling compounds and specific protons situated in the heme pockets of Hb. On the basis of these results, we suggest that one binding site, common to all compounds with anti-sickling activity, is at or near the heme pockets in the alpha and beta chains of both deoxy-HB S and deoxy-Hb A. The proton T1(-1) values of the histidyl residues situated over the surface of the hemoglobin molecule indicate that a second binding site is located at or near the beta 6 position, containing the mutation in Hb S (beta 6Glu----Val). The binding of the compounds investigated to the latter site induces conformational changes in the amino-terminal domains of the beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the aromatic proton magnetic resonance spectrum of crambin

The hydrophobic protein crambin (Mr 4715) has an aromatic content of one phenylalanyl residue (site 13) and two tyrosyl residues (sites 29 and 44). The aromatic residues have been studied by 1H NMR spectroscopy at 300 and 600 MHz for crambin dissolved in deuterated glacial acetic acid and in aqueous organic media. In particular, a 3:1 acetone/water mixture affords a solvent system of low viscosity, which yields very narrow line protein spectra. The aromatic proton spectrum is unusual in that signals are doubled. Spectral simulation of modified and unmodified crambin aromatic spin systems can be accomplished only by assuming that the protein is a mixture of two species. Dynamic 1H-[1H] Overhauser experiments centered on the aromatic doublets of Tyr29 indicate that the two species do not interconvert within 2 s. NMR spectra of crambin samples obtained by repeated crystallization in 17:3 acetone/water indicate that the process enriches the mixture with one component that does not convert to the other, even upon heating of the sample to 351 K. The combined evidence strongly favors the view that the doubled spectrum results from a compositional heterogeneity, most likely a mixture of two homologues, rather than from an equilibrium between interconvertible forms. This is important in view of the fact that its crystallographic structure, solved to 1.5-A resolution [Hendrickson, W.A., & Teeter, M. M. (1981) Nature (London) 290, 107-113], is based on samples containing the two species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of pepsin with aromatic amino acids and their derivatives immobilized to Sepharose

A new LC-MS/MS method for the separation, identification and quantification of residues of 17alpha-estradiol (17alpha-E2) and 17beta-estradiol (17beta-E2) in bovine serum is reported. Deuterium-labelled 17beta-estradiol was used as internal standard. The method was in-house validated in accordance with European Union criteria and adopted in a proficiency study organised by the Community Reference Laboratory (CRL-RIVM, Bilthoven, The Netherlands). The analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a C18 LC column. They were then ionized in a heated nebulizer (HN) interface operating in negative ion mode, where only intact deprotonated molecules, [M-H](-), were generated at m/z 271 and 274 for 17alpha/17beta-E2 and 17beta-E2-d(3), respectively. The decision limits obtained (CCalpha, i.e., critical concentration alpha) were 0.06 ng/mL and 0.03 ng/mL, respectively for 17alpha-E2 and 17beta-E2. Detection capability (CCbeta, i.e., critical concentration beta) values were 0.08 ng/mL and 0.04 ng/mL, respectively, for 17alpha-E2 and 17beta-E2. Precision, accuracy and specificity were satisfactory, recovery ranged from 86.3% to 93.2% and the method resulted sensitive for the required purposes. This method is currently in use for Official Control purposes.     

Evidence for dynamic clustering of carboxy-terminal aromatic amino acids in TonB-dependent energy transduction

Escherichia coli uses the proton motive force of the cytoplasmic membrane and TonB protein to energize the active transport of iron-siderophores and vitamin B12 across the outer membrane. TonB shuttles between the cytoplasmic and outer membranes, presumably during the course of energy transduction. Previous results indicated that the carboxy-terminal 65 amino acids of TonB are essential for both its outer membrane association and activity. A highly conserved region (residues 199-216) within this domain, predicted to be an amphipathic alpha-helix, was the initial focus of this study. Scanning mutagenesis indicated that only the aromatic residues F202, W213 and Y215 were individually important for activity. When the crystal structure of a dimeric TonB carboxy-terminus subsequently became available, we observed that two additional aromatic residues outside that region, F180 and F230, were potentially engaged in end-on hydrophobic interactions with the three residues identified previously. Changing these five aromatic residues individually to alanine reduced TonB activity. Surprisingly, however, each substitution exhibited a unique phenotypic profile with respect to ability to support [55Fe]-ferrichrome transport, sensitivity to colicins B, D, Ia and M or sensitivity to bacteriophage phi80. The phenotypic results suggested that the carboxy-terminus of TonB was a flexible and dynamic domain that could interact specifically with different ligands or transporters, perhaps through the aromatic residues. The possibility of interactions among all the aromatic residues was tested using double-mutant cycle analysis. All possible combinations of alanine substitutions were constructed, with the result that TonB containing any double-alanine substitution was inactive in the phenotypic assays, while retaining the ability to associate with the outer membrane. This synergistic, rather than additive, effect of the double mutants suggested that, consistent with the flexibility suggested by analysis of the single substitutions, all the aromatic residues might be capable of interacting with one another. A means of reconciling these results with the crystal structure is presented.     

Current topics in the biotechnological production of essential amino acids,functional amino acids,and dipeptides

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Chiral nuclear magnetic resonance shift reagents: Lanthanide mixtures with 1-(1-naphthyl) ethylurea derivatives of amino acids

Esters of 1-(1-naphthly)ethylurea derivatives of L-valine, L-leucine, L-tert-leucine, and L-proline are examined as organic-soluble chiral nuclear magnetic resonance (NMR) resolving agents. The reagents are useful for resolving the spectra of chiral sulfoxides, amines, alcohols, and carboxylic acids. Enantiomeric resolution is caused by a combination of diastereomeric effects and the different association constants of the substrates with the resolving agents. Organic-soluble lanthanide species are added to resolving agent-substrate mixtures and often enhance the enantiomeric resolution. The enhancement occurs because the substrate that exhibits weaker binding with the resolving agent is more available to bond to the lanthanide. Broadening in the spectra with lanthanides is reduced at 50°C. Enantiomeric resolution is still observed at elevated temperatures. Chirality 9:1–9, 1997. © 1997 Wiley-Liss, Inc.     

Observation of the phosphatidyl ethanolamine amino proton magnetic resonance in phospholipid vesicles: inside/outside ratios and proton transport.

The amino proton resonance of phosphatidyl ethanolamine in sonicated mixed phospholipid vesicles is observed 3.3 ppm downfield from H₂O. Above pH ～ 5 it is broadened beyond detectability as a result of exchange with H₂O protons. In low salt, resonances of amino protons inside the vesicles appear to persist as the pH is raised, while those on the outside disappear. Solvent catalized proton conduction along the surface is proposed, with an effective -NH₂ to -NH₃ transfer rate of about 8 × 10⁵ sec^?1 at 25°C.     

Drugs for chiral discrimination: Non-coded amino acids and dipeptides by asymmetric hydrogenation

The enantiomers of Propranolol, Pindolol, and Carazolol, well-known β-blockers, have been used to prepare cationic aminophosphine phosphinite rhodium complexes. Propraphos-Rh and Pindophos-Rh are very efficient catalysts in the asymmetric hydrogenation of N-Boc-protected unusual dehydroamino acid derivatives. Carazolol-Rh is less suitable in both activity and enantioselectivity. Under the same conditions, N-Boc-protected dehydrodipeptides are hydrogenated with diastereoselectivities between 70 and 90% de. Chirality 10:535–539, 1998. © 1998 Wiley-Liss, Inc.     

Analysis and identification of aromatic signals in the proton magnetic resonance spectrum of the kringle 4 fragment from human plasminogen

The aromatic 1H NMR spectrum of the kringle 4 domain from human plasminogen has been reexamined in order to identify signals stemming from individual residues. Acid-base titration, nuclear Overhauser effect experiments, and two-dimensional correlated spectroscopies have been implemented in order to analyze the spectrum both in the presence and in the absence of ligands. All six histidyl imidazole singlets have been recognized and paired according to their common side-chain origin. A similar identification has been achieved for the three sets of tryptophanyl resonances, and for Trp-I, the correspondence between indole singlet and multiplets is unambiguously established. The single phenylalanyl side chain and all tyrosyl phenol spin systems have been identified. Titration experiments indicate that one or two of the tryptophans are in the vicinity of carboxyl groups. It is shown that the spectrum for one tyrosyl ring, Tyr-V, undetectable at approximately 300 MHz, becomes visible at 600 MHz, reflecting slow motion on the NMR time scale and a constrained location within the kringle. A simulation of the complete kringle 4 aromatic spectrum is included.     

Side-chain dynamics of two aromatic amino acids in pancreatic phospholipase A2 as studied by deuterium nuclear magnetic resonance

The flexibility of individual amino acid side chains of pancreatic phospholipase A2 in aqueous and micellar solutions was studied with deuterium nuclear magnetic resonance (2H NMR). Bovine pancreatic phospholipase A2 was selectively deuterated at the aromatic ring systems of Trp-3 and Phe-5 and porcine pancreatic phospholipase A2 at Trp-3 only. Solid-state 2H NMR spectra of the lyophilized enzymes exhibited quadrupole splittings on the order of 130 kHz, indicating almost complete immobilization of the aromatic ring systems. Exposure to a water-saturated atmosphere did not remove these steric constraints. However, side-chain mobility could be induced for the tryptophyl residue of the bovine enzyme by dissolving this enzyme in aqueous buffer or micellar solution whereas the phenyl ring always remained immobile and served as a probe for the protein's overall rotation. Typical correlation times for the tryptophyl and phenyl aromatic ring systems in aqueous solution were 7 ps and 13 ns (at 20 degrees C), respectively. The correlation time of the phenyl ring was longer than expected for the monomeric protein (approximately 6 ns), suggesting some aggregation of the protein at the high concentrations used for the NMR measurements. Addition of a micellar solution of oleoylphosphocholine had no influence on the motional freedom of the tryptophyl residue but approximately doubled the correlation time of the phenyl ring, indicating an increase of the effective volume of the tumbling particle due to lipid-protein interaction. A different behavior was observed for the Trp-3 residue of porcine phospholipase A2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear magnetic resonance analyses of side chain conformations of histidine and aromatic amino acid derivatives. Solvent and pH dependence

Stereoselectively beta-deuterated species were synthesized of Ac-His-NHMe, Ac-His-OEt, Ac-His-OH and H-His-NHMe, which are useful as models of histidine residues in peptides. From the spectral comparison of 1H n.m.r., the beta-proton resonances of the normal species were unambiguously assigned. In (C2H3)2SO, C2(2)H5O2H, C2H3O2H, and C5(2)H5N solution and in aqueous solution, the lower-field and higher-field components of beta-proton resonances of the four histidine derivatives are assigned to the pro-R and pro-S protons, respectively. The alternative assignments apply for Ac-His-NHMe, Ac-His-OEt and Ac-His-OH in non-polar solvents such as C2HCl3. Vicinal coupling constants 3J alpha beta S and 3J alpha beta R were obtained for calculating the fractional populations of rotamers about the C alpha-C beta bond. The rotamer populations depend little on the ionization states of the alpha-amino and carboxyl groups or the imidazole ring. The rotamer populations depend significantly on the solvent polarity, similar to those of Phe, Tyr and Trp derivatives. For the two beta-proton resonances of His, Phe, Tyr, and Trp derivatives in a variety of solvents, linear relationships are found between the differences in chemical shifts and the differences in vicinal coupling constants.     

A proton magnetic resonance study of the 18-hydroxy derivatives of cortisol and corticosterone

Proton magnetic resonance spectroscopy played a crucial role in the identification of a new corticosteroid, 18-hydroxycortisol, recently isolated from the urine of a patient with an aldosterone-producing adenoma. Mass spectrometric analysis and chemical degradative studies demonstrated an empirical formula of C21 H30 O6 corresponding to a diketopregnenetetrol and placed three of the four hydroxyl groups at the 11 beta, 18 and 21 positions. The first suggestion that the locus of the fourth hydroxyl was 17 alpha came from the n.m.r. spectrum in the form of negative evidence for proton linked to the carbon atom bearing this fourth hydroxyl. The n.m.r. spectral features of the 18-hydroxy derivatives of cortisol and corticosterone are compared. Both were found to exist in the 20, 18 cyclic hemiketal form.