Biomedical subjects

M M Harding

Publications and source records attributed to M M Harding.

35 records · Page 2Linked to original sources

PubMed1995

Evaluation of Reflection Intensities for the Components of Multiple Laue Diffraction Spots. III. Using a Real-Space Density Modification Method.

In the Laue diffraction pattern, 10--20% of the spots result from the exact superposition of two or more reflections that are ;harmonics', e.g. hkl; 2h 2k 2l, ...; a high proportion of these are low-resolution reflections. For the solution of large or difficult structure problems, the intensities of the remaining 80-90% of the reflections, measurable as singles, may not be sufficient and thus the evaluation of the intensities of the components of the multiple spots is important. A new procedure, DECONV, for this deconvolution using real-space density modifications on the Patterson map is given. This development is based on a procedure in reciprocal space related to direct methods [Hao, Campbell, Harding & Helliwell (1993). Acta Cryst. A49, 528-531]. A Patterson map is calculated using single reflections and modified by removing negative densities and squaring with an appropriate adjustment at the origin peak. The procedure can be repeated until convergence is reached. It has been tested with Laue diffraction data from 4Zn insulin and cytochrome c peroxidase (CCP). 304 and 1134 reflection intensities were evaluated from multiple spots of insulin and CCP, respectively; the mean fractional differences (on F), showing the agreement with high-quality monochromatic data, were 0.27 and 0.21, respectively.

Journal Article↗

PubMed1994

Organometallic anticancer agents: the effect of the central metal and halide ligands on the interaction of metallocene dihalides Cp2MX2 with nucleic acid constituents.

The interactions of the metallocene dihalides Cp2MX2 (M = Ti, Mo, Zr, Hf) and Cp2TiX2 (X = F, Cl, Br, I) and the nucleic acid building blocks D-ribose-5'-phosphate, nucleobases, nucleosides, and nucleotides have been studied by 1H and 31P NMR spectroscopy. In the series Cp2TiX2 (X = F, Cl, Br, I), similar 1H NMR spectra were obtained in titrations of each metallocene with the four nucleotides. The spectra are consistent with dissociation of the halide ligands to give Cp2-Ti2+(aq), which coordinates to nucleobase (N) and phosphate (O) binding sites. The metal center (Ti, Mo, Zr, Hf) strongly influences the nature and extent of interactions between metallocene dichlorides Cp2MCl2 and DNA subunits. Immediate complexation occurs between nucleotides and the antitumor active metallocenes Cp2MX2 (M = Ti and Mo, 0.25-1.0 equiv). In contrast, formation of discrete complexes between nucleotides and the biologically inactive metallocenes Cp2MCl2 (M = Hf, Zr, 0.25-1.00) is not observed, and instead hydrolysis of the Cp rings to give free cyclopentadiene is the major reaction pathway. The complexes formed between titanocene dihalides and nucleotides are stable for hours at pH 2-5; at higher pH the binding is significantly weakened. These results are in agreement with the observed antitumor properties of the metallocene dihalides and provide support for the hypothesis that DNA-metallocene interactions are a major determinant in the antitumor properties of this class of compounds.

Antineoplastic Agents↗

PubMed1994

Synthetic alpha-helical peptides incorporating intercalators for DNA recognition.

The design and synthesis of a water-soluble 14-residue peptide, in which a quinoline intercalator is attached to the peptide backbone via alkylation of a central cysteine residue, is reported. 600 MHz 1H NMR spectroscopy and circular dichroism indicate that the peptide forms a nascent helix in aqueous solution, ie. an ensemble of turn-like structures over several adjacent residues in the peptide. A large number of sequential dNN(i, i+1) connectivities were observed in NOESY spectra, and titration of trifluoroethanol into a solution of the peptide resulted in the characteristic CD spectrum expected for an alpha-helix. At low DNA concentrations, CD spectroscopy indicates that this helical conformation is stabilized, presumably due to folding of the peptide in the major groove of DNA.

Amino Acid Sequence↗

PubMed1993

Solution conformation of the antitumor drug streptonigrin.

The solution conformation of the antitumor drug streptonigrin in THF-d8 has been determined by dynamic 1H NMR spectroscopy (400 MHz). The major solution conformation agrees with the structure observed in the solid state [Chiu, Y.-Y.; Lipscomb, W. N. J. Am. Chem. Soc. 1975, 97, 2525-30]. Rings A, B, and C are coplanar, with ring C held in place by a hydrogen bond from the amino group on ring C and the pyridyl nitrogen in ring B. This conformation is stable in the range pH 3.9-8.9. At lower pH, the hydrogen bond is disrupted due to protonation of the pyridyl nitrogen in ring B. The major species present at pH 3.9-8.9 and 180 K is the zwitterion 1b (80%). Below 190 K, slow proton transfer between the free acid 1a and the zwitterion 1b is observed on the NMR time scale. Addition of a catalytic amount of base to the solution increases the rate of exchange 1a<-->1b, and only one set of resonances is observed. In CD2Cl2 this proton transfer is not observed. Implications for the structure(s) of metal complexes formed by streptonigrin are discussed.

Hydrogen-Ion Concentration↗

PubMed1993

A 31P NMR study of the interaction of the antitumor active metallocene Cp2MoCl2 with calf thymus DNA.

Treatment of sonicated calf thymus DNA with the antitumor active metallocene Cp2MoCl2 afforded a metallocene-DNA complex which was characterized by 31P NMR spectroscopy. In addition to the resonance for the phosphate backbone (delta-1.6), the spectrum contained 2 signals assigned to a phosphate bound Mo-DNA complex(es) (delta 37.2, 36.5) and a broad signal at delta 6.2 ppm. This result suggests that covalent attachment of the metallocene Cp2MoCl2 occurs via phosphate(O) coordination and is accompanied by local distortion of the DNA backbone. This result supports recent ICP studies with Cp2TiCl2 that have DNA detected DNA-metallocene adducts.

Animals↗

PubMed1993

Protein folding in the absence of the solvent ordering contribution to the hydrophobic interaction.

Despite considerable effort there is no consensus as to what interaction, or set of interactions, provides the dominant force that drives protein folding and specifies folded protein structures. A key thermodynamic observation is that a large drop in heat capacity (delta Cp) usually accompanies folding in water. Various factors may contribute to this effect, especially changes in the structure of the solvent upon exposure of both non-polar and polar groups in the unfolded state. The unfavourable Gibbs free energy of solvating non-polar groups, in particular, is thought to provide a central driving force for folding (the hydrophobic effect) but the role of solvent ordering in this remains a matter of controversy. We report here a series of experiments that show that a protein can fold into its native conformation under conditions where solvent ordering effects are demonstrably negligible. In methanol/water mixtures ubiquitin unfolds reversibly with a delta Cp value that falls close to zero above about 30% (v/v) methanol. We are able to reason, on the basis of these data, that the net contribution to the heat capacity change arising primarily from the protein structure itself is not significant and that contributions from changes in solvent ordering are rendered negligible by the change in composition. Nuclear magnetic resonance measurements, however, indicate that non-polar side-chains do still become exposed to solvent in the denatured state under these conditions. The combination of these results and model compound studies suggests that the elimination of ordering effects is an intrinsic property of the mixed solvent. We can, therefore, conclude that the solvent ordering component of the hydrophobic effect is not an obligatory factor in determining the three-dimensional structure into which the protein will fold.

Animals↗

PubMed1992

NMR studies on YSPTSPSY: implications for the design of DNA bisintercalators.

NMR (600 MHz) studies on YSPTSPSY (1), the heptad repeat unit of RNA polymerase II with an extra tyrosine added to the N-terminus, show that the peptide is partially structured in aqueous solution. Peptide 1 contains two overlapping SPXX sequences and has been reported to bind to DNA by bisintercalation (Suzuki, M. Nature 1990, 344, 562-565). In 90% H2O solution at pH 3.2, the major species, which is present in > 90%, contains Pro3 and Pro6 in the trans conformation. At low temperature (4 degrees C), NOE connectivities are consistent with the presence of beta-turn structures in equilibrium with unfolded forms of the peptide. A strong dNN connectivity between Thr4/Ser5, a d alpha N connectivity between Pro3/Thr4, and a medium-range NOE between Pro3 alpha and Ser5 NH indicate the presence of a beta-turn formed by (i) Ser2-Pro3-Thr4-Ser5. A strong dNN connectivity between Ser7/Tyr8 and weaker dN delta NOE connectivities between Ser2/Pro3 delta and Ser7/Pro6 delta were also detected. The solution conformation of the peptide appears to have a crucial role in determining the interaction of the peptide with DNA, given that only bisintercalation has been reported in DNA-binding studies on 1 (Suzuki, M. Nature 1990, 344, 562-565). On the basis of these results, the peptide unit--SPTSPS--(3) has considerable potential as a structured linker in the preparation of DNA bisintercalators of the general structure Xaa-SPTSPS-Zaa (2) with improved selectivity properties.

Amino Acid Sequence↗

PubMed1991

Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in ubiquitin.

A stable, partially structured state of ubiquitin, the A-state, is formed at pH 2.0 in 60% methanol/40% water at 298 K. Detailed characterization of the structure of this state has been carried out by 2D NMR spectroscopy. Assignment of slowly exchanging amide resonances protected from the solvent in the native and A-state shows that gross structural reorganization of the protein has not occurred and that the A-state contains a subset of the interactions present in the native state (N-state). Vicinal coupling constants and NOESY data show the presence of the first two strands of the five-strand beta-sheet that is present in the native protein and part of the third beta-strand. The hydrophobic face of the beta-sheet in the A-state is covered by a partially structured alpha-helix, tentatively assigned to residues 24-34, that is considerably more flexible than the alpha-helix in the N-state. There is evidence for some fixed side-chain--side-chain interactions between these two units of structure. The turn-rich area of the protein, which contains seven reverse turns and a short piece of 3(10) helix, does not appear to be structured in the A-state and is approaching random coil.

Amino Acid Sequence↗

PubMed1990

DNH deoxyribonucleohelicates: self assembly of oligonucleosidic double-helical metal complexes.

Nucleic acids, because of their key biological role, are prime targets for the design of either analogues that may mimic some of their features or of complementary ligands that may selectively bind to and react with them for regulation or reaction. Whereas there has been much work on the latter topic since the elucidation of the double-helical structure of DNA, comparatively little has been done on structural and/or functional models, probably owing to the lack of self-organizing molecular systems. Here we present a class of artificial systems, the nucleohelicates, which are of interest from both points of view because they combine the double-helical structure of the double-stranded metal complexes, the helicates, with the selective interaction features of nucleic-acid bases. These functionalized species allow the study of structural effects on the formation of the double helix and on the binding to other entities, in particular to nucleic acids.

DNA↗

PubMed1985

The crystal structure of beta-lactamase from Staphylococcus aureus at 0.5 nm resolution.

The preparation, crystallization and low-resolution structure determination of beta-lactamase (EC 3.5.2.6, 'penicillinase') from Staphylococcus aureus is described. The enzyme crystallizes in space group I222 with 1 molecule per asymmetric unit and cell dimensions a = 5.45(1), b = 9.39(1) and c = 13.87(2) nm. The structure was determined at 0.5 nm resolution by using phases calculated from (NH4)2Pt(CN)4 and KAu(CN)2 derivatives. The mean figure of merit mean value of m, for the 1106 reflexions used was 0.70. Difference Fourier syntheses for data collected from crystals soaked in platinum D-methionine and in 6-(4-hydroxy-3,5-di-iodobenzamido)penicilloic acid revealed the likely position of the active site of the enzyme.

Carbohydrates↗

PubMed1981