Biomedical subjects

A H Elcock

Publications and source records attributed to A H Elcock.

6 recordsLinked to original sources

PubMed1996

Evidence for electrostatic channeling in a fusion protein of malate dehydrogenase and citrate synthase.

Brownian dynamics simulations were performed to investigate a possible role for electrostatic channeling in transferring substrate between two of the enzymes of the citric acid cycle. The diffusion of oxaloacetate from one of the active sites of malate dehydrogenase (MDH) to the active sites of citrate synthase (CS) was simulated in the presence and absence of electrostatic forces using a modeled structure for a MDH-CS fusion protein. In the absence of electrostatic forces, fewer than 1% of substrate molecules leaving the MDH active site are transferred to CS. When electrostatic forces are present at zero ionic strength however, around 45% of substrate molecules are successfully channeled. As expected for an electrostatic mechanism of transfer, increasing the ionic strength in the simulations reduces the calculated transfer efficiency. Even at 150 mM however, the inclusion of electrostatic forces results in an increase in transfer efficiency of more than 1 order of magnitude. The simulations therefore provide evidence for the involvement of electrostatic channeling in guiding substrate transfer between two of the enzymes of the citric acid cycle. Similar effects may operate between other members of the citric acid metabolon.

Binding Sites↗

PubMed1996

Electrostatic channeling in the bifunctional enzyme dihydrofolate reductase-thymidylate synthase.

The bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) carries out two distinct reactions, with the dihydrofolate produced by the TS-catalyzed reaction acting as the substrate for the DHFR-catalyzed reaction. Brownian dynamics simulation techniques were used to investigate the possible role of electrostatics in determining efficient channeling of the substrate, by explicitly simulating substrate diffusion between the two active sites. With a substrate charge of -2, almost all (> 95%) substrate molecules leaving the TS active site reached the DHFR active site at zero ionic strength. Under the same conditions, but in the absence of electrostatic effects, successful channeling was reduced to only around 6%: electrostatic effects therefore appear essential to explain the efficient channeling observed experimentally. The importance of substrate charge, the relative contributions of specific basic residues in the protein, the role played by the second monomer of the dimer in channeling and the effects of changing ionic strength were all investigated. Simulations performed for substrate transfer in the opposite direction suggest that channeling in DHFR-TS is not strongly directional and that the role of electrostatics is perhaps more one of restricting diffusion of the substrate than one of actively guiding it from the TS to the DHFR active site. The results demonstrate that electrostatic channeling can be a highly efficient means of transferring charged substrates between active sites in solvent-exposed environments.

Binding Sites↗

PubMed1996

Theoretical studies of the intercalation of 9-hydroxyellipticine in DNA.

Extensive molecular dynamics (MD) simulations have been used to investigate the intercalative binding of 9-hydroxyellipticine to the DNA oligonucleotide d(ATATATATATAT)2. Four independent simulations differing in the initial orientation of the drug at the intercalation site were carried out, and compared both with each other and a control simulation of the free DNA sequence. The structure of the latter was compared with structures obtained from x-ray crystallography and nmr spectroscopy, as well as the theoretically derived "alternating B-DNA" model [A. Klug et al. (1979), Journal of Molecular Biology, Vol. 131, p. 669]. The alternation of twist angles observed in experimental structures was reproduced in the simulation. All four independent simulations of the drug-DNA intercalation complex converged in placing the pyridine ring of the ellipticine chromophore in the major groove; in one case this involved a 180 degrees rotation of the drug at the intercalation site. At a more detailed level, the drug is seen to be capable of adopting several distinct orientations, each stable over a period of hundreds of pico-seconds. Despite the presence of several polar groups in the drug, however, no direct hydrogen bonding to the DNA occurs; instead, interactions between the methyl groups of the drug and the thymine bases at the intercalation site appear important in determining the orientational preferences of the drug. Comparison of the intercalation complexes with the free DNA sequence shows a degree of unwinding resulting from intercalation, in good agreement with experimental results, but spread over the three central base-pair steps, not confined to the intercalation site itself. Measurements of torsional rigidity indicate only a slight stiffening of the DNA restricted to the immediate site of intercalation. The structures obtained from the MD simulations were used to calculate theoretical CD spectra, with separate simulations giving very different results. This appears to indicate that given an accurate assignment of the main electronic transition dipole moment of the ellipticine chromophore, discrimination of the more realistic binding geometries may be possible. The relative merits of the various drug orientations observed in the simulations are discussed and a perpendicular orientation of the drug at the intercalation site is considered to be the most consistent with experimental data. While the simulations themselves represent a total of over 2 ns, however, the differences apparent between independent runs indicate that longer simulation times will be required before a complete, unequivocal view of DNA intercalation is obtained.

Base Sequence↗

PubMed1993

SUBCUR: visualization of structural differences between DNA duplexes.

A computer program, SUBCUR, is described which permits analysis and rapid identification of geometrical differences and patterns of variance between two DNA duplexes. The program is compatible with the CURVES 3.1 package and allows graphical visualization of the structural differences. Examples are provided which illustrate the applicability of the program in analyzing the different backbone conformations of two helices and the different curvatures of two helices.

Base Sequence↗

PubMed1991

Sequence selective binding to the DNA major groove: tris(1,10-phenanthroline) metal complexes binding to poly(dG-dC) and poly(dA-dT).

Molecular modelling and energy minimisation calculations that incorporate solvent effects have been used to investigate the complexation of delta and lambda-[Ru(1,10-phenanthroline]2+ to DNA. The most stable binding geometry for both enantiomers is one in which a phenanthroline chelate is positioned in the major groove. The chelate is partially inserted between neighbouring base pairs, but is not intercalated. For delta, though not for lambda, a geometry with two chelates in the major groove is only slightly less favourable. Minor groove binding is shown to be no more favourable than external electrostatic binding. The optimised geometries of the DNA/[Ru(1,10-phenanthroline]2+ complexes enable published linear dichroism spectra to be used to determine the percentage of each enantiomer in the two most favourable major groove sites. For delta 57 +/- 15% and for lambda 82 +/- 7% of bound molecules are in the partially inserted site.

Base Sequence↗

PubMed1991

A binding mode of lambda-[tris(1,10-phenanthroline)ruthenium(II)]2+ exhibiting preference for purine-3',5'-pyrimidine sites of DNA.

Molecular mechanics calculations and molecular dynamics simulations have been used to study the binding of the partially inserted major groove complex of Lambda-[Ru(1,10-phenanthroline)3]2+ with DNA. Energy refinements of this complex showed a clear preference for binding at purine-3',5'-pyrimidine sites over pyrimidine-3',5'-purine sites. The basis for this difference is shown to be a slight change in the binding orientation induced by interchanging the purine and pyrimidine bases. This in turn provides for a better secondary interaction with the helix backbone at a point beyond the immediate binding site. It is this secondary interaction that provides the additional energetic stabilisation for complexes formed at purine-3',5'-pyrimidine sites. Molecular dynamics simulations including explicit representation of solvent support these conclusions and provide an insight into the positional stability of the ligand at a particular site. Repuckering of specific deoxyribose rings to the C3'-endo conformation seems to be an important feature of the DNA/ligand complex.

Base Sequence↗