Low dielectric permittivity of water at the membrane interface: effect on the energy coupling mechanism in biological membranes

Protonmotive force (the transmembrane difference in electrochemical potential of protons, ) drives ATP synthesis in bacteria, mitochondria, and chloroplasts. It has remained unsettled whether the entropic (chemical) component of relates to the difference in the proton activity between two bulk water phases (deltapH(B)) or between two membrane surfaces (deltapH(S)). To scrutinize whether deltapH(S) can deviate from deltapH(B), we modeled the behavior of protons at the membrane/water interface. We made use of the surprisingly low dielectric permittivity of interfacial water as determined by O. Teschke, G. Ceotto, and E. F. de Souza (O. Teschke, G. Ceotto, and E. F. de Sousa, 2001, PHYS: Rev. E. 64:011605). Electrostatic calculations revealed a potential barrier in the water phase some 0.5-1 nm away from the membrane surface. The barrier was higher for monovalent anions moving toward the surface (0.2-0.3 eV) than for monovalent cations (0.1-0.15 eV). By solving the Smoluchowski equation for protons spreading away from proton "pumps" at the surface, we found that the barrier could cause an elevation of the proton concentration at the interface. Taking typical values for the density of proton pumps and for their turnover rate, we calculated that a potential barrier of 0.12 eV yielded a steady-state pH(S) of approximately 6.0; the value of pH(S) was independent of pH in the bulk water phase under neutral and alkaline conditions. These results provide a rationale to solve the long-lasting problem of the seemingly insufficient protonmotive force in mesophilic and alkaliphilic bacteria.     

Mechanism of anion-cation selectivity of amphotericin B channels

Summary Zero current potential and conductance of ionic channels formed by polyene antibiotic amphotericin B in a lipid bilayer were studied in various electrolyte solutions. Nonpermeant magnesium and sulphate ions were used to independently vary the concentration of monovalent anions and cations as well as to maintain the high ionic strength of the two solutions separated by the membrane. Under certain conditions the channels select very strongly for anions over cations. They are permeable to small inorganic anions. However, in the absence of these anions the channels are practically impermeable to any cation. In the presence of a permeant anion the contribution of monovalent cations to channel conductance grows with an increase in the anion concentration. The ratio of cation-to-anion permeability coefficients is independent of the membrane potential and cation concentration, but it does depend linearly on the sum of concentrations of a permeant anion in the two solutions. These results are accounted for on the assumption that a cation can enter only an anion-occupied channel to form an ionic pair at the center of the channel. The cation is also assumed to slip past the anion and then to leave the channel for the opposite solution. This model with only few parameters can quantitatively describe the concentration dependences of conductance and zero current potential under various conditions.     

1H NMR studies on the CuA center of nitrous oxide reductase from Pseudomonas stutzeri.

1H NMR spectra of the CuA center of N2OR from Pseudomonas stutzeri, and a mutant enzyme that contains only CuA, were recorded in both H2O- and D2O-buffered solution at pH 7.5. Several sharp, well-resolved hyperfine-shifted 1H NMR signals were observed in the 60 to -10 ppm chemical shift range. Comparison of the native and mutant N2OR spectra recorded in H2O-buffered solutions indicated that several additional signals are present in the native protein spectrum. These signals are attributed to a dinuclear copperII center. At least two of the observed hyperfine-shifted signals associated with the dinuclear center, those at 23.0 and 13.2 ppm, are lost upon replacement of H2O buffer with D2O buffer. These data indicate that at least two histidine residues are ligands of a dinuclear CuII center. Comparison of the mutant N2OR 1H NMR spectra recorded in H2O and D2O indicates that three signals, c (27.5 ppm), e (23.6 ppm), and i (12.4 ppm), are solvent exchangeable. The two most strongly downfield-shifted signals (c and e) are assigned to the two N epsilon 2H (N-H) protons of the coordinated histidine residues, while the remaining exchangeable signal is assigned to a backbone N-H proton in close proximity to the CuA cluster. Signal e was found to decrease in intensity as the temperature was increased, indicating that proton e resides on a more solvent-exposed histidine residue. One-dimensional nOe studies at pH 7.5 allowed the histidine ring protons to be definitively assigned, while the remaining signals were assigned by comparison to previously reported spectra from CuA centers. The temperature dependence of the observed hyperfine-shifted 1H NMR signals of mutant N2OR were recorded over the temperature range of 276-315 K. Both Curie and anti-Curie temperature dependencies are observed for sets of hyperfine-shifted protons. Signals a and h (cysteine protons) follow anti-Curie behavior (contact shift increases with increasing temperatures), while signals b-g, i, and j (histidine protons) follow Curie behavior (contact shift decreases with increasing temperatures). Fits of the temperature dependence of the observed hyperfine-shifted signals provided the energy separation (Delta EL) between the ground (2B3u) and excited (2B2u) states. The temperature data obtained for all of the observed hyperfine-shifted histidine ligand protons provided a Delta EL value of 62 +/- 35 cm-1. The temperature dependence of the observed cysteine C beta H and C alpha H protons (a and h) were fit in a separate experiment providing a Delta EL value of 585 +/- 125 cm-1. The differences between the Delta EL values determined by 1H NMR spectroscopy and those determined by EPR or MCD likely arise from coupling between relatively low-frequency vibrational states and the ground and excited electronic states.     

The application of 1H NMR chemical shift calculations to diastereotopic groups in proteins.

We have calculated chemical shifts for a range of diastereotopic protons in proteins (i.e. methylene protons, and the methyl groups of valine and leucine residues), using a recently optimised method for chemical shift calculation. The calculations are based on crystal structure coordinates, and have been compared with experimental stereospecific assignments. The results indicate that chemical shifts can be used to suggest stereospecific assignments with about 80% probability of being correct, in cases where both the experimental and the calculated chemical shift differences between a pair of diastereotopic protons are greater than 0.3 ppm. Inaccurate calculations are shown to be caused in most cases by differences between crystal and solution structures. Furthermore, chemical shift calculations based on NMR structures are shown to be capable of acting as a further constraint on structure, by limiting the range of side-chain conformations adopted in structures calculated from NMR data.     

Two gamma-bends in the backbone conformation of [D-Ala2]-leucine enkephalin in solution

The solution conformation of [D-Ala2]-leucine enkephalin in its zwitterionic form in DMSO-d6 has been monitored by one- and two-dimensional proton magnetic resonance spectroscopy at 500 MHz. The resonances from the labile amide protons and the nonlabile protons have been assigned from the shift correlated spectroscopy. The chemical shift of the amide and C-alpha protons are found to vary with temperature but in opposite directions, except the C-alpha proton of the terminal tyrosine residue. This behavior has been explained by the shifting of equilibrium between the zwitterionic and neutral forms of the [D-Ala2]-leucine enkephalin and probably conformational changes accompanying temperature variation. The low values of the temperature coefficients of leucine and glycine amide protons indicate that these protons are either intramolecularly hydrogen bonded or solvent shielded. The observation of sequential cross peaks in the nuclear Overhauser effect spectra obtained at various mixing times, tau m (200-900 ms), indicate an extended backbone, which does not corroborate with the presence of a folded structure, i.e., beta-bend type structure. The estimate of interproton distances in conjunction with the low values of temperature coefficients of the leucine and glycine amide protons and vicinal coupling constants 3JHN-C alpha H have been rationalized by the predominance of two gamma-bends in the backbone conformation of [D-Ala2]-leucine enkephalin. The gamma-bend around the D-Ala residue has phi = 80 degrees and psi = 270 degrees, while the one around Phe it has phi = 285 degrees and psi = 90 degrees.     

Proton nuclear magnetic resonance characterization of the aromatic residues in the variant-3 neurotoxin from Centruroides sculpturatus Ewing

The amino acid sequence for the variant-3 (CsE-v3) toxin from the venom of the scorpion Centruroides sculpturatus Ewing contains eight aromatic residues. By use of 2D NMR spectroscopic methods, the resonances from the individual protons (NH, C alpha H, C beta H',H", and the ring) for each of the individual aromatic residues have been completely assigned. The spatial arrangement of the aromatic ring systems with respect to each other has been qualitatively analyzed by 2D-NOESY techniques. The results show that Trp-47, Tyr-4, and Tyr-42 are in close spatial proximity to each other. The NOESY contacts and the ring current induced shifts in the resonances of the individual protons of Tyr-4 and Trp-47 suggest that the aromatic ring planes of these residues are in an orthogonal arrangement. In addition, the spatial proximity of the rings in the pairs Tyr-4, Tyr-58; Tyr-42, Tyr-40; and Tyr-40, Tyr-38 has also been established. A comparison with the published crystal structure suggests that there is a minor rearrangement of the aromatic rings in the solution phase. No 2D-NOESY contacts involving Phe-44 and Tyr-14 to any other aromatic ring protons have been observed. The pH dependence of the aromatic ring proton chemical shifts has also been studied. These results suggest that the Tyr-58 phenolic group is experiencing a hydrogen-bonding interaction with a positively charged group, while Tyr-4, -14, -38, and -40 are experiencing through-space interactions with proximal negatively charged groups. The Trp-47 indole NH is interacting with the carboxylate groups of two proximal acidic residues. These studies define the microenvironment of the aromatic residues in the variant-3 neurotoxin in aqueous solution.     

The Non-Steady-State Membrane Potential of Ion Exchangers with Fixed Sites

A system of equations, based upon the assumption that the only force acting on each ionic species is due to the gradient of its electrochemical potential, is used to deduce, in the non-steady state for zero net current, the expression of the difference of electric potential between two solutions separated by an ion exchange membrane with fixed monovalent sites. The membrane is assumed to be solely permeable to cations or anions, depending on whether the charge of the sites is -1 or +1, and not to permit any flow of solvent. Under the assumptions that the difference of standard chemical potentials of any pair of permeant monovalent species and the ratio of their mobilities are constant throughout the membrane, even when the spacing of sites is variable, explicit expressions are derived for the diffusion potential and total membrane potential as functions of time and of solution activities. The expressions are valid for any number of permeant monovalent species having ideal behavior and for two permeant monovalent species having “n-type” non-ideal behavior. The results show that for a step change in solution composition the observable potential across a membrane having fixed, but not necessarily uniformly spaced, sites becomes independent of time once equilibria are established at the boundaries of the membrane and attains its steady-state value even while the ionic concentration profiles and the electric potential profile within the membrane are changing with time.     

Heteroassociation of antibiotic norfloxacin with aromatic vitamins in aqueous solution

The heteroassociation of the antibacterial antibiotic norfloxacin with aromatic vitamins nicotineamide and flavin mononucleotide in aqueous solution has been studied by 1H NMR spectroscopy (503 MHz). Equilibrium constants, induced proton chemical shifts, and the thermodynamic parameters (deltaH, deltaS) of the heteroassociation of molecules were determined from the concentration and temperature dependences of chemical shifts of protons of interacting aromatic molecules. An analysis of the results indicates the formation of heterocomplexes between the molecules of the vitamins and norfloxacin, which is caused by stacking interactions between aromatic chromophores and an additional intermolecular hydrogen bond in the norfloxacin-nicotinamide system. Based on the analysis of induced chemical shifts of protons of molecules, the most probable spatial structures 1:1 of norfloxacin-flavin mononucleitide and norfloxacin-nicotinamide heterocomolexes were determined by the methods of molecular modeling using the X-PLOR program.     

Isolation and partial characterization of antagonistic peptides produced by Paenibacillus sp. strain B2 isolated from the sorghum mycorrhizosphere

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of sorghum colonized by Glomus mosseae, produces an antagonistic factor. This factor has a broad spectrum of activity against gram-positive and gram-negative bacteria and also against fungi. The antagonistic factor was isolated from the bacterial culture medium and purified by cation-exchange, reverse-phase, and size exclusion chromatography. The purified factor could be separated into three active compounds following characterization by amino acid analysis and by combined reverse-phase chromatography and mass spectrometry (liquid chromatography-mass spectrometry and mass spectrometry-mass spectrometry). The first compound had the same retention time as polymyxin B1, whereas the two other compounds were more hydrophobic. The molecular masses of the latter compounds are 1,184.7 and 1,202.7 Da, respectively, and their structure is similar to that of polymyxin B1, with a cyclic heptapeptide moiety attached to a tripeptide side chain and a fatty acyl residue. They both contain threonine, phenylalanine, leucine, and 2,4-diaminobutyric acid residues. The peptide with a molecular mass of 1,184.7 contains a 2,3-didehydrobutyrine residue with a molecular mass of 101 Da replacing a threonine at the A2 position of the polymyxin side chain. This modification could explain the broader range of antagonistic activity of this peptide compared to that of polymyxin B.     

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)     

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.     

1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A

The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37 degrees C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results. Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues. The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.     

Use of analogs of primary metabolites in the selection of the producer of polymyxin B]

A number of amino acids were found to have effects on the growth of the polymyxin B-producing culture and biosynthesis of the antibiotic by it. Of special importance was the stimulating effect by methionine. Four selection stages were carried out with using structural analogs of purines and amino acids as selective factors. There were no stable variants with increased antibiotic productivity among the mutants resistant to the analogs of purines and leucine. The levels of polymyxin B accumulation by the variants resistant to 4-fluorophenylalanine were 30 to 50 per cent higher than those in the controls and the variants were characterized by low morphological and antibiotic production variation in the subcultures. The mechanisms of the methionine physiological effect and the prospects of using analogs of the primary metabolites in improvement of the culture producing polymyxin B are discussed.     

Detoxification of amitriptyline by oligochitosan derivatives

Oligo-chitosans were chemically modified with dinitrophenyl groups for selective and rapid adsorption of amitriptyline by forming pi-pi complexes. 1H-NMR was utilized not only for characterization of modified chitosans but also for monitoring the aromatic-aromatic interaction. The variation in the chemical shift of aromatic protons was followed to monitor the aromatic-aromatic interaction. Upfield shift of aromatic protons of dinitrophenyl groups supports aromatic-aromatic interactions with amitriptyline. Drug uptake test by HPLC reveals that dinitrophenyl chitosan particles (1-2 microm) at 0.4 wt% (w/v) in a saline solution (pH 6.9) adsorb 90% amitriptyline within 30 min.     

NMR studies of the trp repressor from Escherichia coli. Characterisation and assignments of residue types

High-resolution proton nuclear magnetic resonance spectra of the trp repressor of Escherichia coli under various conditions are reported and analysed. The spectrum of the denatured state agrees with that predicted from the amino acid composition, with the exception of the two histidine residues, which have different chemical shifts although they titrate normally. The spectrum of the native protein shows the presence of extensive secondary and tertiary structure. Using information from chemical shifts, numbers of protons, titration behaviour, homonuclear chemical-shift-correlated spectroscopy and nuclear Overhauser enhancement correlated spectroscopy, most of the aromatic protons have been assigned to residue type. Further, about 30% of the aliphatic protons have been assigned to residue type by two-dimensional spectroscopy. Nuclear Overhauser enhancements establish that high-field methyl groups belonging to a valine residue lie directly over an aromatic ring.     

Measurement of net proton-hydroxyl permeability of large unilamellar liposomes with the fluorescent pH probe, 9-aminoacridine

The fluorescent probe 9-aminoacridine was used to measure the rate of decay of experimentally established pH gradients across liposome membranes. From the rate of decay, separate permeability coefficients for protons (PH) and hydroxyls (POH) were calculated and summed to yield the net proton-hydroxyl permeability (Pnet). The net permeability of protons and hydroxyls was found to be approximately 10(-4) cm/s, six orders of magnitude greater than that measured for sodium and pyrophosphate ions under similar conditions. This suggests that protons and/or hydroxyls cross lipid bilayers by a different mechanism than do other monovalent cations and anions. In addition, the measurements provide a standard for net proton-hydroxyl permeability in pure phospholipid bilayers for comparison with biological membranes.     

Ring current effects in Adriamycin-flavin mononucleotide complexation as observed by 1H FT NMR spectroscopy

The addition of Adriamycin to a solution containing flavin mononucleotide (FMN) resulted in an upfield shift in the signals of the aromatic ring protons H(6,9) and the 8α, 7α methyl protons of FMN. The chemical shift of the H(6,9) and of the 8α and 7α methyl proton signals of FMN decreased from 7.92, 2.56 and 2.46 ppm, respectively, in the absence of Adriamycin to 7.61, 2.42 and 2.36 ppm, respectively, at 3 mM Adriamycin. Concomitant increases in the linewidth of aromatic and methyl proton siqnals of FMN were also observed. Variable temperature studies over the range of 5 to 43° showed an increase in the chemical shift of both the aromatic and aliphatic proton signals with increasing temperatures. These results suggest that FMN and Adriamycin form a complex via ring-ring stacking.     

Proton nuclear magnetic resonance study of the B9(Asp) mutant of human insulin. Sequential assignment and secondary structure

The sequence-specific 1H nuclear magnetic resonance (n.m.r.) assignment of 49 of the 51 amino acid residues of human B9(Asp) insulin in water at low pH is reported. Spin systems were identified using a series of two-dimensional n.m.r. techniques. For the majority of the amino acid residues with unique spin systems, particularly Ala, Thr, Val, Leu, Ile and Lys, the complete spin systems were identified. Sequence-specific assignments were obtained from sequential nuclear Overhauser enhancement (NOE) connectivities. The results indicate that the solution structure of the mutant closely resembles the crystal structure of native insulin. Thus, the NOE data reveal three helical domains all consistent with the secondary structure of the native human 2Zn insulin in the crystal phase. Numerous slowly exchanging amide protons support these structural elements, and indicate a relatively stable structure of the protein. A corresponding resemblance of the tertiary structures in the two phases is also suggested by slowly exchanging amide protons, and by the extreme chemical shift values observed for the beta-protons of B15(Leu) that agree with a close contact between this residue and the aromatic rings of B24(Phe) and B26(Tyr), as found in the crystal structure of the 2Zn insulin. Finally, there are clear indications that the B9(Asp) insulin mutant exists primarily as a dimer under the given conditions.     

Effect of monovalent anions on the mechanism of phenol hydroxylase

The mechanism of phenol hydroxylase (EC 1.14.13.7) has been studied by steady state and rapid reaction kinetic techniques. Both techniques give results consistent with the Bi Uni Uni Bi ping-pong mechanism proposed for other flavin-containing aromatic hydroxylases. The enzyme binds phenolic substrate and NADPH in that order, followed by reduction of the flavin and release of NADP+. A transient charge transfer complex between reduced enzyme and NADP+ can be detected. Molecular oxygen then reacts with the reduced enzyme-substrate complex. Two to three flavin-oxygen intermediates can be detected in the oxidative half-reaction depending on the substrate, provided monovalent anions are present. Oxygen transfer is complete with the formation of the second intermediate. Based on its UV absorption spectrum and on the fact that oxygen transfer has taken place, the last of these intermediates is presumably the flavin C(4a)-hydroxide. Monovalent anions are uncompetitive inhibitors of phenol hydroxylase. The mechanistic step most affected is the dehydration of the flavin C(4a)-hydroxide to give oxidized enzyme. Chloride also kinetically stabilizes the blue flavin semiquinone of phenol hydroxylase during photoreduction. These data suggest binding of monovalent anions results in stabilization of a proton on the N(5) position of the flavin.     

NMR studies of the interaction of chromomycin A3 with small DNA duplexes I

1H and 31P NMR spectral analysis of a chromomycin/d(ATGCAT)2 complex provides strong evidence for a nonintercalative mode of drug binding. Investigation of the imino proton region of the duplex suggests a protection of one of the two guanine imino protons from fast exchange with the bulk water up to at least 45 degrees C by the drug. Subsequent one-dimensional nuclear Overhauser enhancement experiments place the exchangeable chromomycin chromophoric hydroxyl proton less than 0.45 nm from this guanine imino proton and the chromophore 7-methyl less than 0.45 from the internal thymine 6-proton and/or the guanine 8-proton. 1H two-dimensional NMR reveals that the duplex retains a right-handed B conformation but there are distortions at the TGC region of one chain and large deviations in the chemical shift of protons relative to the uncomplexed duplex in the other chain in the same TGC region. The data suggest that the chromomycin chromophore is oriented such that the hydrophilic side of the ring system is proximal to the helix center in the major groove near the TG region while the aromatic side of the ring is oriented away from the helix but is partially protected from the solvent by the aliphatic chain, which bends back over the two aromatic protons. Changes in the 31P spectrum of the duplex on binding of the drug are different from the effect of either actinomycin or netropsin on nucleic acid fragments.