Insight into the molecular recognition of spermine by DNA quadruplexes from an NMR study of the association of spermine with the thrombin-binding aptamer.

The preferred residence sites and the conformation of DNA-bound polyamines are central to understanding the regulatory roles of polyamines. To this end, we have used a series of selective (13)C-edited and selective total correlation spectroscopy-edited one-dimensional (1D) nuclear Overhauser effect spectroscopy NMR experiments to determine a number of intramolecular (1)H nuclear Overhauser effect (NOE) connectivities in (13)C-labelled spermine bound to the thrombin-binding aptamer. The results provide evidence that the aptamer-bound spermine adopts a conformation that optimizes electrostatic and hydrogen bond contacts with the aptamer backbone. The distance between the nitrogen atoms of the central aminobutyl is reduced by an increase in the population of gauche conformers at the C6-C7 bonds, which results in either a curved or S-shaped spermine conformation. Molecular modelling contributes insight toward the mode of spermine binding of these spermine structures within the narrow grooves of DNA quadruplexes. In each case, the N5 ammonium group makes hydrogen bonds with two nearby phosphates across the narrow groove. Our results have implications for the understanding of chromatin structure and the rational design of quadruplex-binding drugs.

The three-dimensional structure of the 4:1 mithramycin:d(ACCCGGGT)(2) complex: evidence for an interaction between the E saccharides.

Mithramycin and chromomycin, two antitumor drugs, each having an identical aglycone and nearly identical disaccharide and trisaccharide side chains, have differing binding properties to a small oligonucleotide, d(ACCCGGGT)(2) (M. A. Keniry et al., Journal of Molecular Biology, 1993, Vol. 231, pp. 753-767). In order to understand the forces that induce four mithramycin molecules to bind to d(ACCCGGGT)(2) instead of two drug molecules in the case of chromomycin, the structure of the 4:2:1 mithramycin: Mg(2+):d(ACCCGGGT)(2) complex was investigated by (1)H-nmr and restrained molecular dynamics. The resulting three-dimensional model showed that in order to accommodate the close approach of one neighboring mithramycin dimer, the inwardly directed CDE saccharide chain of the neighboring mithramycin dimer undergoes a conformational change such that the E saccharide no longer spans the minor groove but reorients so that the hydrophilic face of the E saccharides from the two dimers oppose each other. Two hydrogen bonds are formed between the hydroxyl groups of the two opposing E saccharide groups. The results are interpreted in terms of the differences in stereochemistry and functional group substitutions between mithramycin and chromomycin. A mithramycin dimer is able to self-associate on an oligonucleotide template because it has two hydroxyl groups on the same face of its terminal E saccharide. A chromomycin dimer is unable to self-associate because one of these hydroxyl groups is acetylated and the neighboring hydroxyl group has a stereochemistry that cannot permit close contact of the hydroxyl group with a neighbouring chromomycin dimer.

The contribution of thymine-thymine interactions to the stability of folded dimeric quadruplexes.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.

Solution structure of the Na+ form of the dimeric guanine quadruplex [d(G3T4G3)]2.

The solution structure of the DNA quadruplex formed by the association of two strands of the DNA oligonucleotide, d(G3T4G3), in NaCl solution has been determined by 1H two-dimensional NMR techniques, full relaxation matrix calculations and restrained molecular dynamics. The refined structure incorporates the sequences 5'-G1sG2AG3AT4AT5AT6AT7AG8sG9AG10A-3' and 5'-G11sG12AG13AT14AT15AT16AT17AG18sG19sG20A-3' (where S and A denote syn and anti, respectively) in a three-quartet, diagonal-looped structure that we [Strahan, G. D., Shafer, R. H. & Keniry, M. A. (1994) Nucleic Acids Res. 22, 5447-5455] and others [Smith, F. W., Lau, F. W. & Feigon, J. (1994) Proc. Natl. Acad. Sci. USA 91, 10546-10550] have described. The loop structure is compact and incorporates many of the features found in duplex hairpin loops including base stacking, intraloop hydrogen bonding and extensive van der Waals' interactions. The first and third loop thymines stack over the outermost G-quartet and are also associated by hydrogen bonding. The second and the fourth loop thymines fold inwards in order to enhance van der Waals' interactions. The unexpected sequential syn-syn deoxyguanosines in the quadruplex stem appear to be a direct consequence of the way DNA oligonucleotides fold and the subsequent search for the most stable loop structure. The implications of loop sequence and length on the structure of quadruplexes are discussed.

The solution conformation of a trisdecanucleotide containing the consensus binding site of the dnaA initiation protein.

The solution structure of a trisdecanucleotide, d(CCTGTGGATAACA).d(TGTTATCCACAGG) containing the consensus binding site of the dnaA initiation protein has been determined by two-dimensional NMR techniques and restrained molecular dynamics calculations. Interproton distances were obtained by an iterative complete relaxation matrix algorithm, MARDIGRAS. During molecular dynamics runs, the backbone was restricted with the assistance of experimentally derived distance constraints. A family of refined structures with small pairwise root-mean-square deviation values (approximately 0.08 nm) was obtained. All but one of the pyrimidines were found to adopt the C1'-exo conformation while the purines were found to adopt the C2'-endo or C1'-exo conformation. The six-membered rings of the purines were found to stack over the six-membered rings of the pyrimidines while there is virtually no overlap of the pyrimidines over the purines. 5'-purine-purine-3' and 5'-pyrimidine-pyrimidine-3' stacking resembles the observed stacking of these bases in other NMR and X-ray structures of oligonucleotides. The final refined structure exhibited a small curvature and was slightly longer than canonical B-DNA. The variation of twist angle, proposed as a recognition element for proteins, exhibited symmetry about the centre of the consensus binding site.

Effect of thermodynamic nonideality in kinetic studies: evidence for reversible unfolding of urease during urea hydrolysis.

A combination of enzyme kinetic studies and active enzyme gel chromatography on Sepharose CL-6B was used to explore conformational changes of the enzyme urease as it catalyzes the hydrolysis of urea in 0.7 M phosphate buffer, pH 7.0, at 20 degrees C. It is shown that elucidation of this system is only possible by studying the effects of inert space-filling macromolecules (ovalbumin and bovine serum albumin) on enzymatic behavior. The resulting increases in reaction velocity are interpreted in terms of composition-dependent activity coefficients assessed on a statistical mechanical basis of excluded volume. The results are first considered in terms of two extreme models; one involving a volume change on the isomerization of the enzyme-substrate complex to its activated state, and the other an isomeric expansion of the enzyme-substrate complex to an inactive form. Although both extreme models provide satisfactory descriptions of the kinetic results, they lead to unrealistic values for the radii of the various states of the enzyme-substrate complex. It is concluded, therefore, that the two isomeric transitions act conjointly, a result in conformity with the previously postulated conformational change associated with formation of the activated enzyme-substrate complex [L. W. Nichol, M. J. Sculley, L. D. Ward, and D. J. Winzor (1983) Arch. Biochem. Biophys. 222, 574-581], and also with the well-established action of the substrate, urea, as an unfolding agent of proteins.

A macromolecular shape function based on sedimentation velocity parameters.

A volume-independent shape function, experimentally determinable from sedimentation velocity experiments, was formulated explicitly in terms of the axial ratio of a macromolecule considered as an ellipsoid of revolution. Use of this function offers a simple first approach to the elucidation of macromolecular geometry as illustrated by calculations for ovalbumin, bovine serum albumin, and myosin.

Physicochemical characterization of high-molecular-weight alpha-crystallin subpopulations from the calf lens nucleus.

Calf lens nuclear alpha-crystallin was separated into five molecular weight subpopulations by exclusion chromatography on Bio-Gel A-5m. These subpopulations were compared by amino acid analysis, ultraviolet absorption analysis, fluorescence, far- and near-ultraviolet circular dichroism, isoelectric focusing, SDS-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Although only minor differences were detectable in most physicochemical properties, progressive changes were found in the near-ultraviolet circular dichroism spectra and in pellet hardness after centrifugation. Minute amounts of beta-crystallin polypeptides and a 43 kDa component were present in all five subpopulations. In addition, the highest molecular weight aggregates contain some gamma-crystallin polypeptides. A slow re-equilibration of separated subpopulations towards the initial distribution was observed by rechromatography.

Structural homology of lens crystallins. II. Homology expressed by correlation coefficients and hydropathy profiles.

Bovine lens alpha A- and alpha B-crystallin polypeptides show extensive sequence homology with each other, but apparently none with beta Bp- and gamma 2-crystallin. Despite only 30% sequence homology, the latter two proteins are assumed to have a strong correspondence in tertiary structure, consisting of four structurally similar folding units of antiparallel beta-sheet. We have tested for internal structural repeats in all crystallins, and structural homology between crystallins, by comparing various physical properties of the amino acid residues, such as bulkiness and propensity to form beta-sheet and beta-turn structure. Two procedures used a combination of five physical parameters to calculate correlation coefficients. The 4-fold structural repeat in gamma 2-crystallin and the internal duplication in beta Bp-crystallin were readily detectable, as was also the strong structural homology between corresponding folding units in beta Bp- and gamma 2-crystallin. However, for alpha-crystallin polypeptides, no conclusive support was obtained for either a four-unit or a six-unit folding, the two models previously considered by us. The third procedure compared smoothened hydropathy plots, representing hydrophilic and hydrophobic regions along the polypeptide sequences. Hydropathy profiles were found to show strong correspondence, particularly between alpha B-crystallin and beta Bp-crystallin. These observations support a similar 4-fold folding pattern for all bovine crystallins. A possible role in subunit interactions of the N-terminal folding unit, which has hydrophobic surface characteristics in both alpha- and beta-crystallin polypeptides, is proposed.

Interactions of lens proteins. Self-association and mixed-association studies of bovine alpha-crystallin and gamma-crystallin.

Concentrated solutions of calf alpha-crystallin (up to 45 g/l) and gamma-crystallin (up to 67 g/l) were subjected to frontal exclusion chromatography at pH 7.3, ionic strength 0.17 and 20 degrees C. The experimental concentration dependence of the weight-average partition coefficient was compared with theoretical expressions, which include considerations of thermodynamic non-ideality effects, for the concentration dependence of a single solute and of a solute undergoing reversible self-association. Two types of association pattern were examined, discrete dimerization and indefinite self-association. The partition chromatography results are consistent with an indefinite self-association of gamma-crystallin, governed by an isodesmic association constant of 6.7 X 10(-3) l/g. alpha-Crystallin appears to self-associate either very weakly, with a maximal association constant of 0.9 X 10(-3) l/g, or not at all; the distinction depends on the assessment of the non-ideality coefficients. The consequences of excluded volume effects on these self-association equilibria at high total protein concentration are discussed. Mixtures of alpha-crystallin and gamma-crystallin were analyzed by frontal exclusion chromatography (up to 14 g/l) and sedimentation velocity (up to 115 g/l): no interaction was observed.

Immunochemical properties of vertebrate alpha-crystallins.

A competitive radioimmunoassay was used to determine the reactivities of alpha-crystallins from 13 species with antibodies directed toward calf alpha-crystallin. The results indicate that species as diverse as human and dogfish share the same number of crossreacting antigenic determinants. The various alpha-crystallins can be distinguished only on the basis of their differing affinities for the antiserum. Hydrophilicity profiles for alpha A and alpha B polypeptides of all species were found to be remarkably similar. On the basis of these, four major sequential determinants could be predicted for each polypeptide. The location and sequence of these determinants were found to be essentially conserved in all alpha-crystallins examined. These results are in agreement with the observed crossreactivities. However, there was little obvious correlation between substitutions in determinants and observed variations in respective alpha-crystallin/antibody affinities. Conservation of antigenic determinants over such a wide evolutionary range may reflect stringent constraints on the overall surface and three-dimensional structure of vertebrate alpha-crystallins.