Ultraviolet resonance Raman spectroscopy of distamycin complexes with poly(dA)-poly(dT) and poly(dA-dT): role of H-bonding

Raman spectra are reported for distamycin, excited at 320 nm, in resonance with the first strong absorption band of the chromophore. Qualitative band assignments to pyrrole ring and amide modes are made on the basis of frequency shifts observed in D2O. When distamycin is dissolved in dimethyl sulfoxide or dimethylformamide, large (30 cm-1) upshifts are seen for the band assigned to amide I, while amides II and III shift down appreciably. Similar but smaller shifts are seen when distamycin is bound to poly(dA-dT) and poly(dA)-poly(dT). Examination of literature data for N-methylacetamide in various solvents shows that the amide I frequencies correlate well with solvent acceptor number but poorly with solvent donor number. This behavior implies that acceptor interactions with the C = O group are more important than donor interactions with the N-H group in polarizing the amide bond and stabilizing the zwitterionic resonance form. The resonance Raman spectra therefore imply that the distamycin C = O groups, despite being exposed to solvent, are less strongly H-bonded in the polynucleotide complexes than in aqueous distamycin, perhaps because of orienting influences of the nearby backbone phosphate groups. In this respect, the poly(dA-dT) and poly(dA)-poly(dT) complexes are the same, showing the same RR frequencies. Resonance Raman spectra were also obtained at 200-nm excitation, where modes of the DNA residues are enhanced. The spectra were essentially the same with and without distamycin, except for a perceptable narrowing of the adenine modes of poly(dA-dT), suggesting a reduction in conformational flexibility of the polymer upon drug binding.     

Raman spectroscopic studies of NAD coenzymes bound to malate dehydrogenases by difference techniques

We report here on the Raman spectra of NADH, 3-acetylpyridine adenine dinucleotide, APAD+, and a fragment of these molecules, adenosine 5'-diphosphate ribose (ADPR) bound to the mitochondrial (mMDH) and cytoplasmic (or soluble, sMDH) forms of malate dehydrogenase. We observe changes in the Raman spectrum of the adenosine moiety of these cofactors upon binding to mMDH, indicating that the binding site is hydrophobic. On the other hand, there is little change in the spectrum of the adenosine moiety when it binds to sMDH. Such observations are in clear contrast with those results obtained in LDH and LADH, where there are significant changes in the spectrum of the adenosine moiety when it binds to these two proteins. A strong hydrogen bond is postulated to exist between amide carbonyl group of NAD+ and the enzyme in the binary complexes with both mMDH and sMDH on the basis of a sizable decrease in the frequency of the carbonyl double bond. The interaction energy for formation of a hydrogen bond is the same as found previously for LDH, and we estimate that it is 2.8 kcal/mol more favorable in the binary complex than in water. A hydrogen bond is also detected between the amide-NH2 group of NADH and sMDH that is stronger than that formed in water and is of the same size as found in LDH. Surprisingly, the hydrogen bond to the -NH2 group in mMDH is the same as that found for water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterodimeric molecules including nucleic acid bases and 9-aminoacridine. Spectroscopic studies, conformations, and interactions with DNA

Heterodimeric molecules have been examined in which the 9-amino-6-chloro-2-methoxyacridine ring is linked to the nucleic acid bases adenine, thymine, and guanine by polymethylenic chains (CH2)n of varying length (n = 3, 5, 6). A detailed analysis has been performed, including hypochromism measurement in the UV, chemical shift variations in Fourier transform proton magnetic resonance, and fluorescence emission. All these techniques show that all molecules exist mainly under folded conformations in water in the temperature range 0-90 degrees C, with the acridine and the base rings being stacked one on top of the other. The thermodynamic parameters for the folded in equilibrium unfolded conformational equilibrium were estimated. The geometry of the intramolecular complexes could be determined. (1) All these data give information on the strength and nature of the base-acridine interactions as a function of different parameters such as solvent, temperature, etc. (2) The molecules under study constitute "spectroscopic models" for the drug-base complexes as they occur in DNA. In particular, we show the dramatic influence of the relative orientations of the two stacked chromophores in the complex upon the magnitude of % H, the percent hypochromism. The quenching and enhancement of acridine fluorescence emission induced respectively by guanine and adenine is evidenced and quantitatively estimated. (3) These base-acridine heterodimers bind to DNA to an extent that is inversely proportional to their degree of intramolecular stacking.     

A common active site model for catalysis by chorismate mutase--prephenate dehydrogenase.

Comparison of the calculated structures for the transition states of the two reactions catalysed by chorismate mutase prephenate dehydrogenase suggests that both reactions could be catalysed at a common active site. Kinetic data for the enzyme from Aerobacteraerogenes are consistent with this possibility. On the basis of these theoretical and experimental data a model for a common active site is developed. In the model, the transition state for each reaction is bound to the enzyme via both of the two substrate carboxyl groups, and can also interact with the coenzyme nicotinamide adenine dinucleotide through a hydrogen bond between the amide moiety of the nicotinamide ring and the hydroxyl group of the substrate. Chorismate, prephenate and 4-hydroxyphenylpyruvate in their ground states form the same hydrogen bond to the coenzyme, but are bound to the enzyme via a single carboxyl group only. The additional bond formed between the enzyme and the transition state structures thus provides the transition state stabilization required for catalysis of both reactions.     

The Analysis of Participation of Individual Proteins in the Protein Interactome Formation

It becomes increasingly clear that most proteins of living systems exist as components of various protein complexes rather than individual molecules. The use of various proteomic techniques significantly extended our knowledge not only about functioning of individual complexes but also formed a basis for systemic analysis of protein-protein interactions. In this study gel-filtration chromatography accompanied by mass spectrometry was used for the interactome analysis of human liver proteins. In six fractions (with average molecular masses of 45 kDa, 60 kDa, 85 kDa, 150 kDa, 250 kDa, and 440 kDa) 797 proteins were identified. In dependence of their distribution profiles in the fractions, these proteins could be subdivided into four groups: (1) single monomeric proteins that are not involved in formation of stable protein complexes; (2) proteins existing as homodimers or heterodimers with comparable partners; (3) proteins that are partially exist as monomers and partially as components of protein complexes; (4) proteins that do not exist in the monomolecular state, but also exist within protein complexes containing three or more subunits. Application of this approach to known isatin-binding proteins resulted in identification of proteins involved in formation of the homo- and heterodimers and mixed protein complexes.     

Actinomycin D complexes with oligonucleotides as models for the binding of the drug to DNA. Paramagnetic induced relaxation experiments on drug-nucleic acid complexes.

Mn(II) ions have been used as a paramagnetic probe to investigate the geometry of drug-oligonucleotide complexes. Nuclear magnetic resonance and electron spin resonance experiments show that Mn(II) ions bind approximately two orders of magnitude stronger to the 5'-terminal phosphate group than to the 3'-5' phosphodiester linkage of deoxydinucleotides. By using mixtures of nucleotides in which only one nucleotide contains a terminal phosphate group, the location of the Mn(II) ion in the drug-nucleotide-Mn(II) complexes may be preselected. The paramagnetic induced relaxation of the nuclear spin systems in these complexes has been used to investigate the geometry of these complexes. These data confirm that actinomycin D is able to recognize and preferentially bind guanine (as opposed to adenine) nucleotides in the quinoid portion of the phenoxazone ring, while both adenine and guanine will bind to the benzenoid portion of the phenoxazone ring. These results suggest that stacking forces are primarily responsible for the general requirement of a guanine base when actinomycin D binds to DNA.     

Solution equilibria and structural characterisation of the palladium(II) and mixed metal complexes of peptides containing methionyl residues

Palladium(II) complexes of the peptides GlyMet, GlyMetGly and GlyGlyMet containing methionyl residues were studied by potentiometric and 1H NMR spectroscopic methods. The coordination of terminal amino and deprotonated amide nitrogen and thioether sulfur donor atoms was suggested in the mono complexes of GlyMet and GlyMetGly. The fourth coordination site of these complexes can be occupied by solvent molecule, chloride or hydroxide ions or by another ligand molecule in the bis or mixed ligand complexes. The second ligand coordinates monodentately via the thioether function in acidic media and the amino group under neutral or basic conditions. The stoichiometry of the major species formed in the palladium(II)-GlyGlyMet system is [PdH(-2) L]- and this is coordinated by the amino, two-amide and the thioether donor functions. Thioether bridged mixed metal complexes formed in the reaction of [Pd(dien)]2+ and [Cu(GlyMetH(-1))] or [Ni(GlyMetGlyH(-2))]- also have been detected by spectroscopic techniques.     

How a protein generates a catalytic radical from coenzyme B(12): X-ray structure of a diol-dehydratase-adeninylpentylcobalamin complex

BACKGROUND: Adenosylcobalamin (coenzyme B(12)) serves as a cofactor for enzymatic radical reactions. The adenosyl radical, a catalytic radical in these reactions, is formed by homolysis of the cobalt-carbon bond of the coenzyme, although the mechanism of cleavage of its organometallic bond remains unsolved. RESULTS: We determined the three-dimensional structures of diol dehydratase complexed with adeninylpentylcobalamin and with cyanocobalamin at 1.7 A and 1.9 A resolution, respectively, at cryogenic temperatures. In the adeninylpentylcobalamin complex, the adenine ring is bound parallel to the corrin ring as in the free form and methylmalonyl-CoA-mutase-bound coenzyme, but with the other side facing pyrrole ring C. All of its nitrogen atoms except for N(9) are hydrogen-bonded to mainchain amide oxygen and amide nitrogen atoms, a sidechain hydroxyl group, and a water molecule. As compared with the cyanocobalamin complex, the sidechain of Seralpha224 rotates by 120 degrees to hydrogen bond with N(3) of the adenine ring. CONCLUSIONS: The structure of the adenine-ring-binding site provides a molecular basis for the strict specificity of diol dehydratase for the coenzyme adenosyl group. The superimposition of the structure of the free coenzyme on that of enzyme-bound adeninylpentylcobalamin demonstrated that the tight enzyme-coenzyme interactions at both the cobalamin moiety and adenine ring of the adenosyl group would inevitably lead to cleavage of the cobalt-carbon bond. Rotation of the ribose moiety around the glycosidic linkage makes the 5'-carbon radical accessible to the hydrogen atom of the substrate to be abstracted.     

Ribosome-inactivating lectins from plants

There is a heterogeneous group of plant proteins which are able to enzymatically inactivate ribosomes by depurination of an invariant adenine from the 28 S ribosomal RNA. Some of these proteins are heterodimers having a lectin subunit which is joined by disulfide bond to the enzymatic subunit. Ricin and abrin which are among the most toxic substances known belong to the last group. This review focuses on the structure of the heterodimeric plant ribosome-inactivating proteins, the way of their action on ribosome, biosynthesis, intracellular trafficking, and their possible usage in medicine.     

Intramolecular stacking interactions in ternary copper(II) complexes formed by a heteroaromatic amine and 9-[2-(2-phosphonoethoxy)ethyl]adenine, a relative of the antiviral nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]adenine

The stability constants of the mixed-ligand complexes formed between Cu(Arm)2+, where Arm=2,2'-bipyridine (Bpy) or 1,10-phenanthroline (Phen), and the dianions of 9-[2-(2-phosphonoethoxy)ethyl]adenine (PEEA2-) and (2-phosphonoethoxy)ethane (PEE2-), also known as [2-(2-ethoxy)ethyl]phosphonate, were determined by potentiometric pH titrations in aqueous solution (25 degrees C; I=0.1 M, NaNO3). The ternary Cu(Arm)(PEEA) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO3) species, where R-PO3(2-) represents a phosph(on)ate ligand with a group R that is unable to participate in any kind of interaction within the complexes. The increased stability is attributed to intramolecular stack formation in the Cu(Arm)(PEEA) complexes and also, to a smaller extent, to the formation of 6-membered chelates involving the ether oxygen atom present in the -CH2-O-CH2-CH2-PO3(2-) residue of PEEA2-. This latter interaction is separately quantified by studying the ternary Cu(Arm)(PEE) complexes which can form the 6-membered chelates but where no intramolecular ligand-ligand stacking is possible. Application of these results allows a quantitative analysis of the intramolecular equilibria involving three structurally different Cu(Arm)(PEEA) species; e.g., of the Cu(Bpy)(PEEA) system about 11% exist with the metal ion solely coordinated to the phosphonate group, 4% as a 6-membered chelate involving the ether oxygen atom of the -CH2-O-CH2CH2-PO3(2-) residue, and 85% with an intramolecular stack between the adenine moiety of PEEA2- and the aromatic rings of Bpy. In addition, the Cu(Arm)(PEEA) complexes may be protonated, leading to Cu(Arm)(H;PEEA)+ species for which it is concluded that the proton is located at the phosphonate group and that the complexes are mainly formed (50 and 70%) by a stacking adduct between Cu(Arm)2+ and the adenine residue of H(PEEA)-. Finally, the stacking properties of adenosine 5'-monophosphate (AMP2-), of the dianion of 9-[2-(phophonomethoxy)ethyl]adenine (PMEA2-) and of several of its analogues (=PA2-) are compared in their ternary Cu(Arm)(AMP) and Cu(Arm)(PA) systems. Conclusions regarding the antiviral properties of several acyclic nucleoside phosphonates are shortly discussed.     

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.     

Three-dimensional structure of the complexes of ribonuclease A with 2',5'-CpA and 3',5'-d(CpA) in aqueous solution, as obtained by NMR and restrained molecular dynamics.

The three-dimensional structure of the complexes of ribonuclease A with cytidyl-2',5'-adenosine (2',5'-CpA) and deoxycytidyl-3',5'-deoxyadenosine [3',5'-d(CpA)] in aqueous solution has been determined by 1H NMR methods in combination with restrained molecular dynamics calculations. Twenty-three intermolecular NOE cross-corrections for the 3',5'-d(CpA) complex and 19 for the 2',5'-CpA, together with about 1,000 intramolecular NOEs assigned for each complex, were translated into distance constraints and used in the calculation. No significant changes in the global structure of the enzyme occur upon complex formation. The side chains of His 12, Thr 45, His 119, and the amide backbone group of Phe 120 are involved directly in the binding of the ligands at the active site. The conformation of the two bases is anti in the two complexes, but differs from the crystal structure in the conformation of the two sugar rings in 3',5'-d(CpA), shown to be in the S-type region, as deduced from an analysis of couplings between the ribose protons. His 119 is found in the two complexes in only one conformation, corresponding to position A in the free protein. Side chains of Asn 67, Gln 69, Asn 71, and Glu 111 from transient hydrogen bonds with the adenine base, showing the existence of a pronounced flexibility of these enzyme side chains at the binding site of the downstream adenine. All other general features on the structures coincide clearly with those observed in the crystal state.     

Intramolecular hydride transfer of a combined coenzyme-substrate analog by D- and L-lactate dehydrogenases

The synthesis of 3-[(3-carboxy-3-oxopropyl)aminocarbonyl]pyridine adenine dinucleotide, a new combined analog of NADH and pyruvate with pyruvate covalently attached to the amide nitrogen atom of the dihydronicotinamide ring via an additional methylene group, is described. In the presence of D-lactate dehydrogenase from Limulus polyphemus, from Lactobacillus leichmannii, and L-lactate dehydrogenase from pig skeletal muscle a redox reaction takes place between the pyruvate moiety and the dihydropyridine ring of the analog. This reaction is shown to be intramolecular by competition experiments with pyruvate. Degradation of the reaction products reveals that the carbon-2 atom of the formed lactate side chain exhibits D configuration in each of these cases studied.     

RNase T1 mutant Glu46Gln binds the inhibitors 2'GMP and 2'AMP at the 3' subsite.

On the basis of molecular dynamics and free-energy perturbation approaches, the Glu46Gln (E46Q) mutation in the guanine-specific ribonuclease T1 (RNase T1) was predicted to render the enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the structures of its two complexes with 2'AMP and 2'GMP. The ribonuclease E46Q mutant is nearly inactive towards dinucleoside phosphate substrates but shows 17% residual activity towards RNA. It binds 2'AMP and 2'GMP equally well with dissociation constants of 49 microM and 37 microM, in contrast to the wild-type enzyme, which strongly discriminates between these two nucleotides, yielding dissociation constants of 36 microM and 0.6 microM. These data suggest that the E46Q mutant binds the nucleotides not to the specific recognition site but to the subsite at His92. This was confirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the Phe100 N and O atoms, and tightly anchored in this position. This interaction may either have locked the guanine recognition site so that 2'AMP and 2'GMP are unable to insert, or the contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable to function in recognition of either guanine and adenine.     

Determination of the interaction of ADP and dADP with copper(II), manganese(II) and lanthanide(III) ions by nuclear-magnetic-resonance spectroscopy.

The aqueous solution conformation of the 1:1 complexes of ADP and dADP bound to a lanthanide ion have been determined by examination of the dipolar shifts and induced relaxation at pH 6.4. Apparent inconsistencies in the observed data are interpreted in terms of a gradually changing lanthanide-oxygen bond length from Pr3+ to Yb3+. The conformations of ADP and dADP are very similar showing an extended diphosphate, a 2E ribose conformation and with the adenine base displaying a small syn contribution. Relaxation data obtained from Mn(II) titrations are readily interpreted in terms of bidentate coordination to alpha and beta phosphates with the nucleotides retaining the same overall conformation found from the lanthanide study. No evidence to support an intramolecular water-bridged backbound structure is observed. The interaction with Cu(II) is more complex, coordination is observed not only at the diphosphate but also at two sites on the base, N-1, and the chelate formed between N-7 and the amino group. The relative importance of these sites is different for ADP and dADP and is also pH-dependent for ADP.     

Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tag.

Soluble guanylyl cyclase (sGC) is an alpha/beta-heterodimeric hemoprotein that, upon interaction with the intercellular messenger molecule NO, generates cGMP. Although the related family of particulate guanylyl cyclases (pGCs) forms active homodimeric complexes, it is not known whether homodimerization of sGC subunits occurs. We report here the expression in Sf9 cells of glutathione S-transferase-tagged recombinant human sGCalpha1 and beta1 subunits, applying a novel and rapid purification method based on GSH-Sepharose affinity chromatography. Surprisingly, in intact Sf9 cells, both homodimeric GSTalpha/alpha and GSTbeta/beta complexes were formed that were catalytically inactive. Upon coexpression of the respective complementary subunits, GSTalpha/beta or GSTbeta/alpha heterodimers were preferentially formed, whereas homodimers were still detectable. When subunits were mixed after expression, e.g. GSTbeta and beta or GSTalpha and beta, no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility of a physiological equilibrium between homo- and heterodimeric sGC complexes.     

Molecular dynamics simulation studies of a protein-RNA complex with a selectively modified binding interface

The RNA recognition motif (RRM) is one of the most common RNA binding domains. We have investigated the contribution of three highly conserved aromatic amino acids to RNA binding by the N-terminal RRM of the U1A protein. Recently, we synthesized a modified base (A-4CPh) in which a phenyl group is tethered to adenine using a linker of 4 methylene groups. The substitution of this base for adenine in the target RNA selectively stabilizes the complex formed with a U1A protein in which one of the conserved aromatic amino acids is replaced with Ala (Phe56Ala). In this article, we report molecular dynamics (MD) simulations that probe the structural consequences of the substitution of A-4CPh for adenine in the wild type and Phe56Ala U1A-RNA complexes and in the free RNA. The simulations suggest that A-4CPh stabilizes the complex formed with Phe56Ala by adopting a folded conformation in which the tethered phenyl group fills the site occupied by the phenyl group of Phe56 in the wild-type complex. In contrast, an extended conformation of A-4CPh is predicted to be most stable in the complex formed with the wild-type protein. The calculations indicate A-4CPh is in an extended conformation in the free RNA. Therefore, preorganizing the structure of the phenyl-tethered base for binding may improve both the affinity and specificity of the RNA containing A-4CPh for the Phe56Ala U1A protein. Taken together, the previous experimental work and the calculations reported here suggest a general design strategy for altering RRM-RNA complex stability.