Structural polymorphism in a tubercidin analogue of the DNA double helix.

A high-angle X-ray fibre diffraction study of a tubercidin analogue of the poly[d(A-T)].poly[d(A-T)] DNA double helix has been carried out using station 7.2 at the Daresbury Laboratory synchrotron radiation source. The polymer has been studied for a wide range of salt strengths and hydration conditions and exhibits conformational polymorphism that is quite distinct from that observed for the unmodified polymer. The replacement of deoxyadenosine by deoxytubercidin in the polynucleotide causes only slight alterations to the structure of A-DNA, but significantly alters the structure of the B conformation. Additionally, the modified polymer does not, in any conditions yet identified, adopt the D conformation. In conditions which would normally favour the D conformation of poly[d(A-T)].poly[d(A-T)], the modified polymer adopts an unusual conformation which is designated here as the K conformation. These observations are important for an understanding of major groove interactions involved in the stabilisation of particular DNA conformations and also more generally for an insight into the pharmacological activity of tubercidin which following its incorporation into nucleic acids may cause stereochemical distortions of the DNA double helix.

Myosin head configuration in relaxed fish muscle: resting state myosin heads must swing axially by up to 150 A or turn upside down to reach rigor.

The arrangement and shape of myosin heads in relaxed muscle have been determined by analysis of low-angle X-ray diffraction data from a very highly ordered vertebrate muscle in bony fish. This reveals the arrangement and interactions between the two heads of the same myosin molecule, the shape of the resting myosin head (M.ADP.Pi) assuming a putative hinge between the myosin catalytic domain and the light chain binding-domain, and the way that the actin-binding sites on myosin are arrayed around the actin filaments in the bony fish muscle A-band cell unit. The results are discussed in terms of possible force-generating mechanisms. Changes in myosin head shape or tilt have been implicated in the mechanism of force generation. The myosin head arrangement, including perturbations from perfect helical symmetry, has all heads oriented roughly the same way up (there is only a small range of rotations around the head long axis). X-ray data do not define the absolute polarity of the myosin head array. The resting head rotation is either similar to (65 degrees difference) or opposite to (115 degrees difference) the rotation in the rigor state. If the rotations are similar, probably the more likely possibility, then the average relative axial displacement of the inner and outer ends of the heads from the resting state to rigor is about 140 to 150 A. If (less likely) the resting head rotation is opposite to rigor, then the heads would need to turn over (i.e. rotate about 115 degrees around their own long axes) and the mean relative axial displacement from relaxed to rigor would only be 20 to 30 A.

A high-angle neutron fibre diffraction study of the hydration of deuterated A-DNA.

A high-angle neutron fibre diffraction study of the hydration of A-DNA has been performed using the single-crystal diffractometer D19 at the Institut Laue-Langevin (Grenoble, France). The sample was prepared using deuterated DNA extracted from E. Coli cells cultured on deuterated nutrients. In common with our previous neutron fibre diffraction studies of DNA, this work exploits the ability to isotopically replace H2O around the DNA by D2O. However this study benefitted additionally from the fact that the hydrogen atoms which are covalently bonded to carbon atoms in the DNA sugars and bases were replaced by deuterium so that incoherent scattering and absorption effects were minimised. Successive cycles of Fourier synthesis and Fourier difference synthesis allowed water peaks to be identified and their positional and occupancy parameters to be refined against the observed diffraction data. The results confirm the main hydration features noted in our earlier studies with a clear network of water running along the inside edge of the major groove linking successive OI phosphate oxygen atoms. The central core of water running along the axis of the double helix is very much clearer in this work. Additionally this study shows chains of ordered water lying in the centre of the major groove.

Structure determination of OppA at 2.3 A resolution using multiple-wavelength anomalous dispersion methods.

OppA is a 58.8 kDa bacterial transport protein involved in the transport of peptides across the cytoplasmic membrane of Gram-negative bacteria. It binds peptides from two to five residues in length but with little sequence specificity. OppA from Salmonella typhimurium has been cloned and expressed in E. coli and the protein cocrystallized with uranyl acetate, producing two distinct crystal forms with different uranium sites. Multiple-wavelength data collected about the uranium L(III) edge have been collected at the Daresbury Synchrotron Radiation Source (SRS) to a nominal resolution limit of 2.3 A. Maximum-likelihood phasing methods have been used in phase determination from the multiple-wavelength data giving a readily interpretable electron-density map, without any density modification. The electron-density map, calculated at 2.3 A resolution shows OppA to be a bilobal, principally beta-stranded, three-domain protein. The tri-lysine ligand molecule can be clearly seen in the peptide-binding site between the two lobes.