Structure of a DNA intercalation complex as determined by NMR using a paramagnetic probe.

Longitudinal relaxation rates of the protons of the 3,8-dimethyl-N-methyl-phenanthrolinium (DMP) cation in solutions containing DNA are strongly affected by the addition of the paramagnetic manganese (II) ions due to the electron-nuclear dipolar interaction in the ternary Mn-DNA-DMP Complex. Two possible models for the DMP-DNA intercalation complex are examined and one of them is unequivocally discriminated on the basis of the proton relaxation data. It is concluded that in the intercalation complex the long axis of the DMP molecule is almost perpendicular to the hydrogen bonds of the DNA base-pairs.     

Dodecylphosphocholine micelles as a membrane-like environment: new results from NMR relaxation and paramagnetic relaxation enhancement analysis

To further examine to what extent a dodecylphosphocholine (DPC) micelle mimics a phosphatidylcholine bilayer environment, we performed ¹³C, ²H, and ³¹P NMR relaxation measurements. Our data show that the dynamic behavior of DPC phosphocholine groups at low temperature (12 °C) corresponds to that of a phosphatidylcholine interface at high temperature (51 °C). In the presence of helical peptides, a PMP1 fragment, or an annexin fragment, the DPC local dynamics are not affected whereas the DPC aggregation number is increased to match an appropriate area/volume ratio for accommodating the bound peptides. We also show that quantitative measurements of paramagnetic relaxation enhancements induced by small amounts of spin-labeled phospholipids on peptide proton signals provide a meaningful insight on the location of both PMP1 and annexin fragments in DPC micelles. The paramagnetic contributions to the relaxation were extracted from intra-residue cross-peaks of NOESY spectra for both peptides. The location of each peptide in the micelles was found consistent with the corresponding relaxation data. As illustrated by the study of the PMP1 fragment, paramagnetic relaxation data also allow us to supply the missing medium-range NOEs and therefore to complete a standard conformational analysis of peptides in micelles. Received: 16 April 1998 / Revised version: 19 June 1998 / Accepted: 30 July 1998     

Determination of solution structures of paramagnetic proteins by NMR

Standard procedures for using nuclear Overhauser enhancements (NOE) between protons to generate structures for diamagnetic proteins in solution from NMR data may be supplemented by using dipolar shifts if the protein is paramagnetic. This is advantageous since the electron-nuclear dipolar coupling provides relatively long-range geometric information with respect to the paramagnetic centre which complements the short-range distance constraints from NOEs. Several different strategies have been developed to date, but none of these attempts to combine data from NOEs and dipolar shifts in the initial stages of structure calculation or to determine three dimensional protein structures together with their magnetic properties. This work shows that the magnetic and atomic structures are highly correlated and that it is important to have additional constraints both to provide starting parameters for the magnetic properties and to improve the definition of the best fit. Useful parameters can be obtained for haem proteins from Fermi contact shifts; this approach is compared with a new method based on the analysis of dipolar shifts in haem methyl groups with respect to data from horse and tuna ferricytochromes c. The methods developed for using data from NOEs and dipolar shifts have been incorporated in a new computer program, PARADYANA, which is demonstrated in application to a model data set for the sequence of the haem octapeptide known as microperoxidase-8. Received: 13 October 1997 / Accepted: 19 December 1997     

Structure of Leu5-enkephalin bound to a model membrane as determined by high-resolution NMR

Summary Opioid peptides are thought to interact with the cell membrane in their biological journey to the membrane-bound receptor. Both organic solvents and model membranes have been used previously to determine the stable solution conformations of peptide hormones. Leucine enkephalin has been studied in a number of different environments, but with limited resolution. Here it is shown that leucine enkephalin forms a stable type IV -turn structure in dodecylphosphocholine micelles. We have observed a highly solvent-shielded amide proton with no evidence for a complementary hydrogen bond acceptor. The structural details of the peptide as determined by NMR spectroscopy in solution are described.     

Ruthenium red as a paramagnetic probe in NMR spectroscopy

It is shown that ruthenium red acts as a paramagnetic probe in NMR spectroscopy. Unlike lanthanide and calcium ions it acts as a substitution probe for polyamine binding sites in biological systems, although it also binds at sites where calcium binds.     

2D NMR of paramagnetic metalloenzymes: Cyanide-inhibited horseradish peroxidase

Summary Two-dimensional (2D) proton NMR correlation spectroscopy, COSY, and nuclear Overhauser spectroscopy, NOESY, have been used to explore the applicability of these methods for the moderately large (42 KDa), paramagnetic cyanide-inhibited derivative of horseradish peroxidase, HRP-CN. The target resonances are those in the active site of HRP-CN which experience substantial hyperfine shifts and paramagnetic relaxation. The magnitude COSY experiment was found to yield cross peaks for all known spin-coupled heme substituents, as well as for the majority of non-heme hyperfine shifted protons, in spite of line widths of the order of 100 Hz. Moreover, the rapid relaxation of the hyperfine-shifted resonances allows the extremely rapid collection of useful 2D NMR data sets without the loss of information. For the heme, the combination of COSY cross peaks for the vinyl and propionate substituents, and NOESY cross peaks among these substituent protons and heme methyls, allows assignment of heme resonances without recourse to deuterium labeling of the heme. A seven-proton coupled spin system was identified in the upfield region that is consistent with originating from the proposed catalytic Arg³⁸ residue in the distal heme pocket, with orientation relative to the heme similar to that found in cytochromec peroxidase. The upfield hyperfine-shifted methyl group in the substrate binding pocket previously proposed to arise from Leu²³⁷ is shown to arise instead from an as yet unidentified Ile. NOESY spectra collected at very short (3 ms) and intermediate (20 ms) mixing times indicate that build-up curves can be obtained that should yield estimates of distances in the heme cavity. It is concluded that 2D NMR studies should be able to provide the heme assignments, aid in identifying the catalytic residues, and provide information on the spatial disposition of such residues in the active site for cyanide complexes of a number of intermediate to large paramagnetic heme peroxidases, as well as for other paramagnetic metalloenzymes with line widths of 100 Hz. Moreover, paramagnetic-induced hyperfine shifts and linewidths to 100 Hz need not interfere with the complete solution structure determination of a small paramagnetic protein solely on the basis of 2D NMR data.     

Interplay between minor and major groove-binding transcription factors Sox2 and Oct1 in translocation on DNA studied by paramagnetic and diamagnetic NMR

The pathways whereby Sox2 scans DNA to locate its specific binding site are investigated by NMR in specific and nonspecific Sox2·DNA complexes and in a specific ternary complex with Oct1 on the Hoxb1 regulatory element. Direct transfer of Sox2 between nonspecific sites on different DNA molecules occurs without dissociation into free solution at a rate of ～10(6) M(-1) s(-1), whereas one-dimensional sliding proceeds with a diffusion constant of ≥0.1 μm(2)·s(-1). Translocation of Sox2 from one specific DNA site to another occurs via jumping, involving complete dissociation into free solution (k(d) ～5-6 s(-1)) followed by reassociation (k(a) ～5 × 10(8) M(-1) s(-1)). In the presence of Oct1 bound to an adjacent specific site, k(d) is reduced by more than 10-fold. Paramagnetic relaxation measurements, however, demonstrate that sparsely populated (<1%), transient states involving nonspecifically bound Sox2 in rapid exchange with specifically bound Sox2 are sampled in both binary Sox2·DNA- and ternary Oct1·Sox2·Hoxb1-DNA-specific complexes. Moreover, Sox2 modulates the mechanism of translocation of Oct1. Both Sox2 and the Oct1 POU(HD) domain are transiently released from the specific ternary complex by sliding to an adjacent nonspecific site, followed by direct transfer to another DNA molecule, whereas the Oct1 POU(S) domain is fixed to its specific site through direct interactions with Sox2. Intermolecular translocation of POU(HD) results in the formation of a bridged intermediate spanning two DNA molecules, enhancing the probability of complete intermolecular translocation of Oct1. By way of contrast, in the specific Oct1·DNA binary complex, POU(S) undergoes direct intermolecular translocation, whereas POU(HD) scans the DNA by sliding.     

Intermolecular dynamics studied by paramagnetic tagging

Yeast cytochrome c and bovine adrenodoxin form a dynamic electron transfer complex, which is a pure encounter complex. It is demonstrated that the dynamic nature of the interaction can readily be probed by using a rigid lanthanide tag attached to cytochrome c. The tag, Caged Lanthanide NMR Probe 5, induces pseudocontact shifts and residual dipolar couplings and does not perturb the binding interface. Due to the dynamics in the complex, residual dipolar couplings in adrenodoxin are very small. Simulation shows that cytochrome c needs to sample a large part of the surface of adrenodoxin to explain the small degree of alignment observed for adrenodoxin. The applied method provides a simple and straightforward way to observe dynamics in protein complexes or domain–domain mobility without the need for external alignment media. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Probing the surface of a sweet protein: NMR study of MNEI with a paramagnetic probe

The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure-function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis.     

Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR

The geometry of the axial ligands of the hemes in the triheme cytochrome PpcA from Geobacter sulfurreducens was determined in solution for the ferric form using the unambiguous assignment of the NMR signals of the α-substituents of the hemes. The paramagnetic ¹³C shifts of the hemes can be used to define the heme electronic structure, the geometry of the axial ligands, and the magnetic susceptibility tensor. The latter establishes the magnitude and geometrical dependence of the pseudocontact shifts, which are crucial to warrant reliable structural constraints for a detailed structural characterization of this paramagnetic protein in solution.     

The fluorinated anesthetic halothane as a potential NMR biologic probe

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The ¹⁹F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the ¹⁹F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, ¹⁹F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo ¹⁹F-NMR surface coil and imaging studies.     

Equilibria and kinetics of the intercalation of Pt-proflavine and proflavine into calf thymus DNA

The interaction of the cis-platinum derivative of proflavine [[PtCl(tmen)(2)][HNC(13)H(7)(NHCH(2)CH(2))(2)]](+) (PRPt) with CT-DNA is investigated by spectrophotometry and T-jump relaxation in 0.11M NaCl, pH 7.0, and 25 degrees C. The DNA-proflavine (PR) system is investigated under the same conditions. Static measurements indicate that base-dye interactions prevail and their analysis reveals that the site size for PRPt (n=2.6) is twice that found for PR (n=1.3). One relaxation effect is observed for the DNA/PR system and two effects for the DNA/PRPt system, the faster of them being similar to that of DNA/PR. The kinetics of the process are discussed in terms of the three-step sequence D+S <= => DS(I) <= => DS(II) <= => DS(III), where PR and the aromatic residues of PRPt intercalate into DNA by the same mechanism. The third step represents the penetration of platinum residues between base-pairs and is associated to remarkable enthalpy and entropy changes. Further mechanistic details are discussed.     

DNA as a target for antimicrobials

Resistance to antimicrobials is one of the biggest threats to our healthcare. However, in the last few decades very few truly novel antimicrobial compounds have been brought to market, creating the potential threat of a post-antibiotic era in which infections are very difficult to treat. Identification of novel compounds with antimicrobial activity is therefore paramount. Ideally, novel compounds should be designed that are active against targets that are not or barely used, as it is less likely that resistance already exists against such compounds. One example of an underexplored target in the treatment of infections is DNA. In this review we describe a number of DNA binding compounds and discuss potential opportunities and problems.     

Structure determination of a Galectin-3-carbohydrate complex using paramagnetism-based NMR constraints

The determination of the location and conformation of a natural ligand bound to a protein receptor is often a first step in the rational design of molecules that can modulate receptor function. NMR observables, including NOEs, often provide the basis for these determinations. However, when ligands are carbohydrates, interactions mediated by extensive hydrogen-bonding networks often reduce or eliminate NOEs between ligand and protein protons. In these cases, it is useful to look to other distance- and orientation-dependent observables that can constrain the geometry of ligand-protein complexes. Here we illustrate the use of paramagnetism-based NMR constraints, including pseudo-contact shifts (PCS) and field-induced residual dipolar couplings (RDCs). When a paramagnetic center can be attached to the protein, field-induced RDCs and PCS reflect only bound-state properties of the ligand, even when averages over small fractions of bound states and large fractions of free states are observed. The effects can also be observed over a long range, making it possible to attach a paramagnetic center to a remote part of the protein. The system studied here is a Galectin-3-lactose complex. A lanthanide-binding peptide showing minimal flexibility with respect to the protein was integrated into the C terminus of an expression construct for the Galectin-3-carbohydrate-binding domain. Dysprosium ion, which has a large magnetic susceptibility anisotropy, was complexed to the peptide, making it possible to observe both PCSs and field-induced RDCs for the protein and the ligand. The structure determined from these constraints shows agreement with a crystal structure of a Galectin-3-N-acetyllactosamine complex.     

Changes in C NMR chemical shifts of DNA as a tool for monitoring drug interactions

The antibiotic drug, netropsin, was complexed with the DNA oligonucleotide duplex [d(GGTATACC)]₂ to explore the effects of ligand binding on the ¹³C NMR chemical shifts of the DNA base and sugar carbons. The binding mode of netrospin to TA-rich tracts of DNA has been well documented and served as an attractive model system. For the base carbons, four large changes in resonance chemical shifts were observed upon complex formation: −0.64 ppm for carbon 4 of either Ado4 or Ado6, 1.36 ppm for carbon 2 of Thd5, 1.33 ppm for carbon 5 of Thd5 and 0.94 for carbon 6 of Thd5. AdoC4 is covalently bonded to a heteroatom that is hydrogen bonded to netropsin; this relatively large deshielding is consistent with the known hydrogen bond formed at AdoN3. The three large shielding increases are consistent with hydrogen bonds to water in the minor groove being disrupted upon netropsin binding. For the DNA sugar resonances, large changes in chemical shifts were observed upon netropsin complexation. The 2′, 3′ and 5′ ¹³C resonances of Thd3 and Thd5 were shielded whereas those of Ado4 and Ado6 were deshielded; the ¹³C resonances of 1′ and 4′ could not be assigned. These changes are consistent with alteration of the dynamic pseudorotational states occupied by the DNA sugars. A significant alteration in the pseudorotational states of Ado4 or Ado6 must occur as suggested by the large change in chemical shift of −1.65 ppm of the C3′ carbon. In conclusion, ¹³C NMR may serve as a practical tool for analyzing structural changes in DNA-ligand complexes.     

Rubredoxin as a paramagnetic relaxation-inducing probe

The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with unpaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c₃ was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M2¹ and M18¹. The rubredoxin binding surface in the complex with cytochrome c₃ was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c₃ are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in ¹HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes.     

Discrimination between closely related Triticeae species using genomic DNA as a probe

Summary Labelled total genomic DNA was used as a probe in combination with blocking DNA to discriminate between taxonomically closely related species in the genera Hordeum and Secale. Discrimination was possible both by Southern hybridization to size-fractionated restriction enzyme digests of genomic DNA and by in situ hybridization to chromosome preparations. To distinguish between two species (e.g. H. vulgare and H. bulbosum), genomic DNA from one species was used as the labelled probe, while unlabelled DNA from the other species was applied at a much higher concentration as a block. The blocking DNA presumably hybridized to sequences in common between the block and the labelled probe, and between the block and DNA sequences on the membrane or chromosomes in situ. If so, mainly species-specific sequences would remain as sites for probe hybridization. These species-specific sequences are dispersed and represent a substantial proportion of the genome (unlike many cloned, species-specific sequences). Consequently, rapid nonradioactive methods detected probe hybridization sites satisfactorily. The method was able to confirm the parentage of hybrid plants. It has potentially wide application in plant breeding for the detection of alien DNA transfer, and it can be easily adapted to many species.     

HMG-D complexed to a bulge DNA: an NMR model

An NMR model is presented for the structure of HMG-D, one of the DROSOPHILA: counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA(2) DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high-resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA(2) bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non-sequence-specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG-DNA complex structures are discussed.     

Variable role of ions in two drug intercalation complexes of DNA

The crystal structures of the hexamer duplex d(CGTACG)₂ complexed with the intercalating anthraquinone derivative 1,5-bis[3-(diethylamino)propionamido]anthracene-9,10-dione and the acridine derivative 9-acridinyl tetralysine have been solved at 2.0- and 1.4-Å resolution, respectively. In both cases, the drugs adopt multiple orientations within a large DNA cavity constituted by two groups of four approximately coplanar bases. Cations play a pivotal role in the crystal structure. Both complexes crystallise in the presence of Co²⁺, Ba²⁺ and Na⁺ ions. They reveal at least two different types of coordination environments: (1) specific sites for Co²⁺ interacting with N7 of guanine; (2) a central ionic site formed by four phosphate groups, which can be occupied by different ions. One more ionic site that is not always occupied by ions is also visible in the electron density map. All of them play a role in the crystal structure.     

Cytotoxic trans-oriented iminoether platinum complexes - Kinetics of binding to DNA oligonucleotides determined by N NMR spectroscopy

The kinetics of reactions between cytotoxic trans-oriented iminoether platinum complexes and DNA oligonucleotides have been studied by 1D and 2D [¹H, ¹⁵N] HMQC NMR spectroscopy. The results for the two isomers of the mono-iminoether compound trans-[PtCl₂(NH₃){E/Z-HNC(OMe)Me}] (trans-E and trans-Z) are compared with those of the bis-iminoether derivative trans-[PtCl₂{E-HNC(OMe)Me}₂] (trans-EE). Earlier we have shown that quite unexpectedly, trans-EE is practically inert towards a central GG residue in a 12-mer double-helical duplex. We now show that the less bulky trans-E and trans-Z compounds do bind to the interior of the duplex [5′-d(G₁G₂T₃A₄C₅C₆G₇G₈ T₉A₁₀C₁₁C₁₂)]₂ which contains terminal and central “hot” GG site. The platination by trans-E and trans-Z is as expected most pronounced for the solvent exposed, terminal GG-step but significantly, competitive binding is also observed for the central GG-step. The rate of platination of the terminal G-sites is almost an order of magnitude larger for the oligomer than for the monomer GMP which was studied for comparison. The role of trans-platinum carrier ligands in influencing the type and rate of formation of adducts with DNA and other relevant biomolecules is discussed.