Oligochitosan derivatives bearing electron-deficient aromatic rings for adsorption of amitriptyline: implications for drug detoxification

The objective of this work is the synthesis of water-soluble oligochitosan derivatives with electron deficient aromatic rings for selective and rapid adsorption of amitriptyline through pi-pi complexation. Oligochitosan was chemically modified under homogeneous conditions in dimethyl sulfoxide (DMSO). (1)H NMR, FT-IR, and MALDI-TOF were employed in characterization, confirming that the electron deficient aromatic rings were chemically attached to the backbone of oligochitosan. Thromboelastography (TEG) revealed functionalized oligochitosan derivatives did not affect blood clotting. (1)H NMR was also utilized to observe the aromatic-aromatic interaction between electron deficient aromatic rings on oligochitosan and electron rich aromatic rings in amitriptyline. The chemical shift variation of aromatic protons in oligochitosan derivatives was followed to monitor the aromatic-aromatic interaction. Upfield shift of aromatic protons on benzenesulfonyl and dinitrobenzenesulfonyl groups was observed upon the addition of amitriptyline, supporting the formation of pi-pi complexes through aromatic-aromatic interactions. Dinitrobenzenesulfonyl rings show a larger variation in chemical shift due to the presence of the electron deficient nitro groups.     

Probing weakly polar interactions in cytochrome c.

Theoretical, statistical, and model studies suggest that proteins are stabilized by weakly polar attractions between sulfur atoms and properly oriented aromatic rings. The two sulfur-containing amino acids, methionine and cysteine, occur frequently among functional alleles in random mutant libraries of Saccharomyces cerevisiae iso-1-cytochrome c genes at positions that form a weakly polar aromatic-aromatic interaction, the wild-type protein. To determine if a weakly polar sulfur-aromatic interaction replaced the aromatic-aromatic interaction, the structure and stability of two variants were examined. Phenylalanine 10, which interacts with tyrosine 97, was replaced by methionine and cysteine. The cysteine was modified to form the methionine and cysteine analog, S-methyl cysteine (CysSMe). Proton NMR studies indicate that changing Phe 10 to Met or CysSMe affects only local structure and that the structures of sulfur-containing variants are nearly identical. Analysis of chemical shifts and nuclear Overhauser effect data indicates that both sulfur-containing side chains are in position to form a weakly polar interaction with Tyr 97. The F10M and F10CSMe variants are 2-3 kcal mol-1 less stable than iso-1-cytochrome c at 300 K. Comparison of the stabilities of the F10M and F10CSMe variants allows evaluation of the potential weakly polar interaction between the additional sulfur atom of F10CSMe and the aromatic moiety of Tyr 97. The F10CSMe;C102T variant is 0.7 +/- 0.3 kcal mol-1 more stable than the F10M;C102T protein. The increased stability is explained by the difference in hydrophobicity of the sulfur-containing side chains. We conclude that any weakly polar interaction between the additional sulfur and the aromatic ring is too weak to detect or is masked by destabilizing contributions to the free energy of denaturation.     

The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315

Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol. 41, in the press). Light-absorption studies indicate a dinitrophenyl–tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate–tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl–Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H₍₃₎ resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H₍₃₎ proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93_L, in an `aromatic box' of essentially tryptophan-93_L, phenylalanine-34_H and tyrosine-34_L; asparagine-36_L and tyrosine-34_L also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody–hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures.     

Selective modification of myosin SH1 with 1,2,4-trinitrobenzene. VIII. Thiols of myosin

Myosin has 2 mol of the most reactive thiol, named SH1. 1,2,4-Trinitrobenzene (TNB), a novel dinitrophenyl(DNP)ating reagent [Takahashi et al. (1983) Chem. Lett. 1445-1448], was found to react only with SH1 without any other amino acid residues in myosin under the conditions used. Its reaction with myosin SH1 was about 30 times faster than that with N-acetylcysteine (NAC). The reaction rate of TNB with SH1 was about twice compared with that of NEM, the most reactive selective reagent for SH1 so far found, although its rate with NAC was only one sixtieth that of NEM. As to the lambda max of the absorption spectrum of SH1-DNP-myosin, a large red shift of as much as 20 nm was observed compared with low molecular S-DNP derivatives. This red shift disappeared in 8 M urea. This outstanding feature of SH1 modification with TNB was discussed in terms of affinity labeling by interaction with an aromatic amino acid near SH1.     

Comparison of cooperative and isolated site binding of T4 gene 32 protein to ssDNA by 1H NMR

Deuteriation of all aromatic protons of gene 32 protein (g32P) from phage T4, followed by selective introduction of specific protons, has allowed the precise identification of the number and magnitude of the chemical shift changes induced in the aromatic protons when g32P binds noncooperatively or cooperatively to nucleotides. Signals from five Tyr residues are shifted by binding of g32P to d(pA)8 or d(pA)40-60; however, the change from noncooperative, d(pA)8, to cooperative, d(pA)40-60, binding causes significant increases in the magnitudes of the shifts for only two of these Tyr signals. These two Tyr residues may interact directly with the nucleotide bases, while the shifts associated with the other three Tyr may be due to conformational changes in g32P upon ssDNA binding. Similar conclusions can be drawn for two of the six Phe residues whose protons undergo shifts upon nucleotide binding. Observation of selected proton signals allows for the first time detection by 1H NMR of changes in the proton signals from two Trp residues upon nucleotide binding. The side chains of two Tyr, one or two Phe, and one Trp are probably directly involved in nucleotide base-protein interactions. As assayed by the signals from the H2 and H8 protons of adenine, the bases of a bound nucleotide are undergoing a fast chemical exchange in the noncooperative mode of binding, but shift to slow exchange upon assuming the cooperative mode of ssDNA interaction. When bound to a polynucleotide, the A domain of g32P (residues 254-301) becomes more mobile, as reflected in sharpening of the 1H NMR signals from the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR and CD studies on the interaction of Alzheimer beta-amyloid peptide (12-28) with beta-cyclodextrin

Polymerization of the amyloid beta-peptide (Abeta) has been identified as a major feature of the pathogenesis of Alzheimer's disease (AD). Inhibition of the formation of these toxic polymers of Abeta has emerged as an approach for developing therapeutics for AD. NMR and CD spectra were used to investigate the interaction between cyclodextrin and Abeta(12-28) peptide, which was reported to be an important region for forming amyloid fibrils. CD spectral analyses show that of the alpha-, beta- and gamma-cyclodextrins only beta-cyclodextrin inhibits the aggregation of Abeta(12-28) at pH 5.0. Analysis of the one-dimensional proton NMR spectra of Abeta(12-28) and the mixture of Abeta(12-28) with beta-cyclodextrin clearly indicates that there are chemical shift changes in the aromatic ring of Phe19 and the methyl groups of Val18 in the peptide. The NOESY spectra show cross-peaks between H-3 and H-5 of beta-cyclodextrin and the aromatic protons of Phe19 and Phe20. These chemical shift differences and NOEs demonstrate that there is an interaction between Abeta(12-28) and beta-cyclodextrin. Analysis of the cross-peak intensity in the NOESY spectra reveals that the aromatic rings of Phe19 and 20 are generally inserted into beta-cyclodextrin at the broad side and are oriented toward the narrow side of the cavity.     

Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water-miscible organic mixtures

We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.     

Conformation of cobrotoxin in aqueous solution as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of cobrotoxin from Naja naja atra were observed in 2H2O solution. The pKa value (5.93) of His-32 is slightly lower than the pKa value (6.65) of the reference model of N-acetylhistidine methylamide, because of the electrostatic interaction with Arg-33 and Asp-31. The pKa value (5.3--5.4) of His-4 is appreciably low, because of the interaction with the positively charged guanidino group possibly of Arg-59. The hydrogen-deuterium exchange rates in 2H2O solution were measured of cobrotoxin and imidazole-bearing models. The second-order rate constants of N-acetylhistidine methylamide, N-acetylhistidine and imidazole acetic acid satisfy the Br?nsted relation. With reference to this Br?nsted relation, the imidazole ring of His-32 is confirmed to be exposed. The imidazole ring of His-4 is also exposed and the exchange rate is excessively promoted by the presence possibly of Arg-59 in the proximity. All the methyl proton resonances are assigned to amino-acid types, by conventional double-resonance method and more effectively by the spin-echo double-resonance method. Eight methyl proton resonances are identified as due to the gamma and/or delta-methyl groups of Val-46, Leu-1, Ile-50 and Ile-52 residues. The proximity of aromatic ring protons and methyl protons is elucidated by the analyses of nulcear Overhauser effect enhancements. The aromatic proton resonances of Trp-29 are affected by the ionizable groups of Asp-31, His-32 and Tyr-35. The methyl groups of Ile-50 are in the proximity to the aromatic ring of Trp-29 and the methyl groups of Ile-52 are in the proximity to Tyr-25. The highest-field methyl proton resonance is due to a threonine residue in the proximity to His-4. The appreciable temperature-dependent chemical shift of this methyl proton resonance suggests a temperature-dependent local conformational equilibrium around the His-4 residue of the first loop of the cobrotoxin molecule.     

Aromatic-aromatic interactions and protein stability. Investigation by double-mutant cycles

The side-chains of phenylalanine and tyrosine residues in proteins are frequently found to be involved in pairwise interactions. These occur both within repeating elements of secondary structure and in tertiary and quaternary interactions. It has been suggested that they are important in protein folding and stability, and non-bonded potential energy calculations indicate that a typical aromatic-aromatic interaction has an energy of between -1 and -2 kcal/mol and contributes between -0.6 and -1.3 kcal/mol to protein stability. There is such an aromatic pair on the solvent-exposed face of the first alpha-helix of barnase, the small ribonuclease from Bacillus amyloliquefaciens. The edge of the aromatic ring of Tyr17 interacts with the face of that of Tyr13. The two residues have been mutated both singly and pairwise to alanine, and their free energies of unfolding determined by denaturation with urea. Application of the double-mutant cycle analysis gives an interaction energy of -1.3 kcal/mol for the aromatic pair in the folded protein relative to solvation by water in the unfolded protein. This value is similar to that calculated from the change in surface-accessible area between the rings on the formation of the pair. Analysis of a further double-mutant cycle in which the Tyr residues are mutated to Phe indicates that the aromatic-aromatic interactions of Tyr/Tyr and Phe/Phe make identical contributions to protein stability. However, Tyr is preferred to Phe by 0.3(+/- 0.04) kcal/mol at the solvent-exposed face of the alpha-helix.     

Interaction of substrate analogs with bovine pancreatic ribonuclease A as studied by 1H nuclear magnetic resonance

The 270 MHz 1H NMR spectra of 3'-UMP and 3'-CMP were observed in the presence of a two-fold molar excess of bovine pancreatic RNase A [EC 3.1.27.5] at various pHs. For the C(5), C(6), and C(1') protons of these nucleotides, the pH profiles of chemical shifts induced by binding to RNase A were obtained by plotting the differences between chemical shifts in the presence and the absence of RNase A against pH. Such profiles were bell-shaped for the C(5) and C(6) protons of both 3'-UMP and 3'-CMP. However the profiles of C(1') protons were not bell-shaped but appeared to consist of two bell-shaped curves and reflect the dissociations of at least three ionizable groups. The observations for the C(1') protons suggest that there are at least two forms of complexes different from each other in the interaction reflecting the chemical shift of the C(1') proton. In order to clarify the interacting sites of ribonucleotides affecting the induced shift profile of the C(1') proton, the pH titration curves were observed for 3'-dCMP in the presence of RNase A. The induced shift profile was bell-shaped for the C(1') proton as well as for the C(5) proton of the base. This indicates that the interaction at the O(2')H [or O(2')] sites of ribonucleotides causes the two forms of complexes of 3'-UMP and 3'-CMP with RNase A. The interacting sites and modes were discussed with these and the pH titration curves of His-12, His-119, and Phe-120 of RNase A in the presence of a three-fold molar excess of ribonucleotides.     

Contribution of tryptophan residues to the combining site of a monoclonal anti dinitrophenyl spin-label antibody

Two Fab fragments of the monoclonal anti dinitrophenyl (DNP) spin-label antibody AN02 were prepared by recombination of specifically deuterated heavy and light chains. In the recombinant H(I)L(II) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the heavy chain. In the recombinant H(II)L(I) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the light chain. Saturation of three resonances of H(I)L(II), assigned to tryptophan protons of the light chain, resulted in magnetization transfer to the aromatic proton at position 6 of the DNP ring and to the CH2 protons of the glycines linked to the DNP in a diamagnetic hapten (DNP-DG). Saturation of three resonances of H(II)L(I) assigned to tryptophan protons of the heavy chain resulted in magnetization transfer to the CH2 protons of the glycines in DNP-DG. From the dependence of the magnetization transfer on the irradiation time, the cross relaxation rates between the involved protons were estimated. The inferred distances between these protons of the hapten and certain tryptophan protons are 3-4 A. It is concluded that in the combining site of AN02 there is one tryptophan from the light chain and one tryptophan from the heavy chain that are very near the hapten. When all tyrosines and phenylalanines were perdeuterated and all tryptophan aromatic protons were deuterated except for the protons at positions 2 and 5, titration of the Fab fragments with variable amounts of paramagnetic hapten showed that one proton from the light chain tryptophan is near (less than 7 A) the unpaired electron and that three other protons are significantly closer than 15 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interatomic potentials and solvation parameters from protein engineering data for buried residues

Van der Waals (vdW) interaction energies between different atom types, energies of hydrogen bonds (H-bonds), and atomic solvation parameters (ASPs) have been derived from the published thermodynamic stabilities of 106 mutants with available crystal structures by use of an originally designed model for the calculation of free-energy differences. The set of mutants included substitutions of uncharged, inflexible, water-inaccessible residues in alpha-helices and beta-sheets of T4, human, and hen lysozymes and HI ribonuclease. The determined energies of vdW interactions and H-bonds were smaller than in molecular mechanics and followed the "like dissolves like" rule, as expected in condensed media but not in vacuum. The depths of modified Lennard-Jones potentials were -0.34, -0.12, and -0.06 kcal/mole for similar atom types (polar-polar, aromatic-aromatic, and aliphatic-aliphatic interactions, respectively) and -0.10, -0.08, -0.06, -0.02, and nearly 0 kcal/mole for different types (sulfur-polar, sulfur-aromatic, sulfur-aliphatic, aliphatic-aromatic, and carbon-polar, respectively), whereas the depths of H-bond potentials were -1.5 to -1.8 kcal/mole. The obtained solvation parameters, that is, transfer energies from water to the protein interior, were 19, 7, -1, -21, and -66 cal/moleA(2) for aliphatic carbon, aromatic carbon, sulfur, nitrogen, and oxygen, respectively, which is close to the cyclohexane scale for aliphatic and aromatic groups but intermediate between octanol and cyclohexane for others. An analysis of additional replacements at the water-protein interface indicates that vdW interactions between protein atoms are reduced when they occur across water.     

Assignment of aromatic resonances in the 1H nuclear magnetic resonance spectra of cardioactive polypeptides from sea anemones

High-resolution 1H NMR spectroscopy at 300 MHz has been used to investigate the aromatic residues of a series of homologous polypeptides from sea anemones: anthopleurin-A from Anthopleura xanthogrammica and toxins I and II from Anemonia sulcata. Using two-dimensional NMR techniques, specific assignments to individual protons have been made for all aromatic resonances in the spectra of these molecules. In all three polypeptides the resonances from the two conserved Trp residues, 23 and 33, are shifted significantly from their random coil values, and the indole NH resonance of Trp-23 is not observed. These shift perturbations are due in part to a mutual interaction of the two indole rings, which is also indicated by the observation of nuclear Overhauser enhancements between protons of the two rings. Several other nonpolar side chains also interact with these two Trp residues, forming a hydrophobic region, the overall structure of which is conserved throughout the series. The other aromatic residues in these polypeptides appear not to participate in this structural region.     

Aromatic interactions in model peptide β-hairpins: ring current effects on proton chemical shifts

Crystal structures of eight peptide β-hairpins in the sequence Boc-Leu-Phe-Val-Xxx-Yyy-Leu-Phe-Val-OMe revealed that the Phe(2) and Phe(7) aromatic rings are in close spacial proximity, with the centroid-centroid distance (R(cen)) of 4.4-5.4 ? between the two phenyl rings. Proton NMR spectra in chloroform and methanol solution reveal a significant upfield shift of the Phe(7) C(δ,δ') H(2) protons (6.65-7.04 ppm). Specific assignments of the aromatic protons have been carried out in the peptide Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (6). The anticipated ring current shifts have been estimated from the aromatic ring geometrics observed in crystals for all eight peptides. Only one of the C(δ,δ') H proton lies in the shielding zone with rapid ring flipping, resulting in averaging between the two extreme chemical shifts. An approximate estimate of the population of conformations, which resemble crystal state orientation, may be obtained. Key nuclear Overhauser effects (NOEs) between facing Phe side chains provide support for close similarity between the solid state and solution conformation. Temperature dependence of aromatic ring proton chemical shift and line widths for peptide 6 (Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe) and the control peptide Boc-Leu-Val-Val-(D)Pro-Gly-Leu-Phe-Val-OMe establish an enhanced barrier to ring flipping when the two Phe rings are in proximity. Modeling studies suggest that small, conformational adjustment about C(α)-C(β) (χ(1) ) and C(β)-C(γ) (χ(2) ) bonds of both the Phe residues may be required in order to permit unhindered, uncorrelated flipping of both the Phe rings. The maintenance of the specific aromatic ring orientation in organic solvents provides evidence for significant stabilizing interaction.     

The gross architecture of an antibody-combining site as determined by spin-label mapping

1. A series of Dnp (dinitrophenyl) nitroxide spin labels was used to map the dimensions of the combining site of the Dnp-binding immunoglobulin A myeloma protein MOPC 315. The method compares the observed e.s.r. (electron-spin-resonance) hyperfine splittings with those calculated on the basis of different postulated motions for the spin label. The analysis is complicated by the sensitivity of the e.s.r. hyperfine splitting to the overall ;tumbling' time of the antibody-hapten complex and the polarity of the spin-label's environment. When these effects are considered quantitatively, it is then possible to determine the degree of mobility of each hapten which is allowed by the shape of the combining site. 2. The dinitrophenyl ring is rigidly held, and the depth of the site is 1.1-1.2nm and has lateral dimensions at the entrance to the site >/=0.6nmx0.9nm. The analysis of the results for spin-labelled haptens with chiral centres allows these lateral dimensions to be refined to 0.8nm and 1.1nm, and it is shown that the site is asymmetric with respect to the plane of the dinitrophenyl ring. 3. A polarity profile of the combining site was also obtained and a positively charged amino acid residue, possibly arginine-95(L) (light chain), was located at the entrance to the site. 4. The binding of Gd(III) to the antibody-hapten complexes results in quenching of the e.s.r. signal of the nitroxide. By using La(III) as a control, the paramagnetic contribution to the quenching is measured. 5. Analysis of the differential quenchings of the enantiomers of two five-membered nitroxide ring spin labels gives two possible locations of the metal-binding site. One of these is equidistant (0.7nm) from each of the three dinitrophenyl aromatic protons, and nuclear-magnetic-resonance relaxation studies, at 270MHz, on solutions of dinitrobenzene, Gd(III) and the Fv fragment (variable region of heavy and light chain) from protein MOPC 315 support this location for the metal site. 6. The e.s.r. and metal-binding data were then compared with the results of a model of the combining site constructed on the basis of framework invariance in immunoglobulins [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol.41, in the press]. The overall agreement is very good. Assignments of possible chelating groups for the metal can be made.     

1H- and 31P-NMR studies of ditercalinium binding to a d(GCGC)2 and d(CCTATAGG)2 minihelices: a sequence specificity study

The structures of the complexes formed in aqueous solution between ditercalinium, a bis-intercalating drug, and both the self-complementary tetranucleotide d(GCGC)2 and octanucleotide d(CCTATAGG)2, have been investigated by 400-MHz 1H-nmr and 162-MHz 31P-nmr. All the nonexchangeable protons, as well as the exchangeable imino protons and the phosphorus signals, have been assigned. Both oligonucleotides have been shown to adopt a right-handed B-DNA type structure. The addition of ditercalinium to the oligonucleotides lead to the formation of complexes in slow exchange at the nmr time scale with the free helices. At all drug-to-helix ratios studied, the ditercalinium was found in the bound form, whereas free and complexed oligonucleotides were in slow exchange, allowing resonance assignments through two-dimensional chemical exchange experiments. for d(GCGC)2 the strong upfield shifts induced on all aromatic protons of both the bases and the drug by complexation with ditercalinium suggest an interaction by intercalation of the two rings. However, the loss of twofold symmetry upon binding, as well as the chemical shift variation of the drug proton signals of one of the chromophores with temperature and concentration, favor a model in which the drug-nucleotide complexes have one ring of the drug intercalated and the other stacked on top of the external base pair. The intermolecular contacts between drug protons and nucleotide protons give a defined geometry for complexation that is consistent with the proposed model. In contrast, with d(CCTATAGG)2 several drug-nucleotide complexes were formed and a large increase in line broadening was observed at high drug-to-DNA ratios, precluding a detailed analysis of these complexes. However, the large upfield shift in the imino proton resonances together with the shielding of the ditercalinium ring protons favor a model with bis-intercalation of ditercalinium. This model is supported by the downfield shift of at least 4 out of 14 phosphorus signals. The results are compared with those obtained on ditercalinium binding to the homologous sequences d(CGCG)2 and d(TTCGCGAA)2, and discussed in terms of sequence specificity.     

A hydrogen-deuterium exchange study of the amide protons of polymyxin B by nuclear-magnetic-resonance spectroscopy.

1. Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution. 2. Hydrogen-deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid-catalysed reaction more than the monovalent anions. 3. The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the leucine methyl and gamma-CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N-H protons are used to identify two beta turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N-H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity.     

A 500 MHz proton NMR study of stacking interactions: binding of tripeptide Lys-Tyr-Lys to tetradeoxynucleotide d-GpCpGpC.

The complete sequential assignment and conformation of d-GpCpGpC in D2O has been determined from 1D NMR spectra at 285-320 K and room temperature 2D-COSY and NOESY spectra. The tetradeoxynucleotide exists primarily as a right handed double helix at 285 K, having Tm as 314 K. On binding to a tripeptide Lys-Tyr-Lys in a concentration equimolar to tetranucleotide duplex, the Tyr ring protons shift upfield by 0.14 ppm at 285 K. The increase in Tm on binding suggests stabilization of duplex. The existence of intermolecular NOEs between C4 sugar protons and Tyr alpha C and Lys alpha C protons give direct evidence of proximity of Tyr residue to the C4 base of d-GpCpGpC. The conformation of d-GpCpGpC remains unchanged on binding. The observed results are interpreted in terms of preferential stacking of aromatic ring of Tyr residue with proximal base-pair of d-GpCpGpC, stabilized by electrostatic interaction of Lysine side chains with backbone phosphates. This is in contrast to intercalculation of aromatic dyes within base-pairs resulting in a change in sugar conformation at the binding site.     

Novel circular dichroism spectroscopic approach for detection of ligand binding of proteins: Avidin as example

Various globular proteins having low or no α-helical content exhibit atypical far-ultraviolet (UV) circular dichroism (CD) spectra due to aromatic-aromatic residue and aromatic residue-peptide bond exciton coupling interactions. As a representative example of such proteins, far-UV CD spectra of chicken avidin were recorded before and after the addition of different small molecules. Intensity increase-decrease and/or wavelength shift of the positive CD peak of avidin at 228 nm were observed in the presence of various drugs, dyes, and natural compounds. The results were interpreted by exciton interactions between the aromatic residues of the biotin binding site and the substances bound to it. This novel, fast, microgram (μg) scale approach can be applied for detection of ligand binding of additional proteins displaying avidin-like far-UV CD spectra.     

1H NMR (500 MHz) of gene 32 protein--oligonucleotide complexes

In concentrated solutions, gene 32 single-stranded DNA binding protein from bacteriophage T4 (gene 32P) forms oligomers with long rotational correlation times, rendering 1H NMR signals from most of the protons too broad to be detected. Small flexible N- and C-terminal domains are present, however, the protons of which give rise to sharp resonances. If the C-terminal A domain (48 residues) and the N-terminal B domain (21 residues) are removed, the resultant core protein of 232 residues (gene 32P) retains high affinity for ssDNA and remains a monomer in concentrated solution, and most of the proton resonances of the core protein can now be observed. Proton NMR spectra (500 MHz) of gene 32P and its complexes with ApA, d(pA)n (n = 2, 4, 6, 8, and 10), and d(pT)8 show that the resonances of a group of aromatic protons shift upfield upon oligonucleotide binding. Proton difference spectra show that the 1H resonances of at least one Phe, one Trp, and five Tyr residues are involved in the chemical shift changes observed with nucleotide binding. The number of aromatic protons involved and the magnitude of the shifts change with the length of the oligonucleotide until the shifts are only slightly different between the complexes with d(pA)8 and d(pA)10, suggesting that the binding groove accommodates approximately eight nucleotide bases. Many of the aromatic proton NMR shifts observed on oligonucleotide complex formation are similar to those observed for oligonucleotide complex formation with gene 5P of bacteriophage fd, although more aromatic residues are involved in the case of gene 32P.(ABSTRACT TRUNCATED AT 250 WORDS)