Biomedical subjects

J V Skelly

Publications and source records attributed to J V Skelly.

7 recordsLinked to original sources

PubMed1992

Molecular structure of the B-DNA dodecamer d(CGCAAATTTGCG)2. An examination of propeller twist and minor-groove water structure at 2.2 A resolution.

The crystal structure of the dodecanucleotide duplex d(CGCAAATTTGCG)2 has been solved to 2.2 A resolution and refined to an R-factor of 18.1% with the inclusion of 71 water molecules. The structure shows propeller twists of up to -20 degrees for the A.T base-pairs, although there is probably only one (weak) three-centre hydrogen bond in the six base-pair AT narrow minor-groove region. An extensive ribbon of hydration has been located in this groove that has features distinctive from the classic "spine of hydration". Solvation around phosphate groups is described, with several instances of water molecules bridging between phosphates.

Base Composition↗

PubMed1992

Conformational properties of the G.G mismatch in d(CGCGAATTGGCG)2 determined by NMR.

The conformational properties of the DNA duplex d(CGCGAATTGGCG)2, which contains two noncomplementary G.G base pairs, have been examined in aqueous solution by 1H and 31P NMR as a function of temperature. The G.G mismatch is highly destabilizing, with a Tm value 35 K below that observed for the native EcoRI dodecamer. The dodecamer appears symmetric in the NMR spectra and exists largely as an average B-type DNA conformation. However, the 1H and 31P NMR spectra give evidence of considerable conformational heterogeneity at the mismatched nucleotides and their nearest neighbors, which increases with increasing temperature. There is no evidence for a significant population of the syn purine conformation. The imino protons of the mispaired bases G4 and G9 are degenerate, resonate at high field, and exchange readily with solvent. These results indicate that the mispaired bases are only weakly hydrogen-bonded and are only partially stacked into the helix. On raising the temperature, the duplex shows increasing exchange between two or more conformations originating from the mismatch sites. However, these additional conformations maintain their Watson-Crick hydrogen bonding. The increase in chemical exchange is consistent with a quasimelting process for which the G.G sites provide local nuclei. Extensive modeling studies by dynamic annealing have confirmed that the G(anti).G(anti) conformation is favored and that the mispairs are poorly stacked within the helix. The results explain both the poor thermal stability and low hypochromicity of this duplex.

Base Sequence↗

PubMed1990

Conformational effects of nucleotide exchange in ras p21 proteins as studied by fluorescence spectroscopy.

The intrinsic fluorescence properties of the oncogene protein p21N-ras,p21H-ras and one of its transforming mutants, p21N-ras (Val12), have been investigated. A mutant containing a single tryptophan at position 28 in p21H-ras (Trp28) has been specifically engineered to provide a probe of protein conformation on nucleotide binding. The proteins produced essentially similar circular dichroism spectra typical of alpha/beta proteins. A decrease in the intensity of the fluorescence emission spectrum due to tyrosine occurred on GDP/GTP nucleotide exchange in the native and mutant proteins. Selective excitation of the single tryptophan in p21 produced a decrease in fluorescence intensity which was accompanied by a blue shift in the wavelength of maximum emission on nucleotide exchange. A reduction in the residual Mg2+ ion concentration enhanced this effect.

Circular Dichroism↗

PubMed1990

Crystal structure of a berenil-dodecanucleotide complex: the role of water in sequence-specific ligand binding.

The three-dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti-trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5'-AAT-3' region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen-bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen-bonded contact with an amidinium nitrogen atom, N3 of the 5' end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.

Amidines↗

PubMed1990

Crystal structure and sequence-dependent conformation of the A.G mispaired oligonucleotide d(CGCAAGCTGGCG).

The crystal structure of the dodecanucleotide d(CGCAAGCTGGCG) has been determined to a resolution of 2.5 A and refined to an R factor of 19.3% for 1710 reflections. The sequence crystallizes as a B-type double helix, with two G(anti).A(syn) base pairs. These are stabilized by three-center hydrogen bonds to pyrimidines that induce perturbations in base-pair geometry. The central AGCT region of the helix has a wide (greater than 6 A) minor groove.

Adenine↗

PubMed1989

Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line.

An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.

Animals↗

PubMed1979

Platelet counting-development of a reference method and a reference preparation.

A reference method has been defined for platelet counting by counting chamber haemocytometry, and a procedure has been developed, using platelet rich plasma (PRP) and a Coulter ZBI counter which provides a reliable, rapid and relatively simple method for platelet counting which is closely comparable to the reference method and can thus be used both as a secondary reference method and a routine method. The optimal method for obtaining PRP from EDTA blood was by augmented sedimentation with Boyum's methyl cellulose-metrizoate mixture at sp. gr. 1.08. This was shown to yield a platelet suspension which reflects closely the platelet content of the original blood. Calibration of electronic counters for platelet counting requires material which is stable and which parallels natural human platelets in size, distribution and other physical characteristics. A suspension of glutaraldehyde-fixed human platelets in glycerol appears to be suitable as a reference preparation. Its method of production is described.

Calibration↗