John H. Venable

The structure of ψ DNA

Abstract In concentrated solutions of neutral or anionic polymers (and an adequate cation concentration), DNA condenses into a compact state, which is of interest for its possible relevance to chromosome structure and the packing of DNA in viruses. The X-ray scattering of DNA condensed in this way has been examined with respect to the secondary structure of the helix and the tertiary structure of the compact state. Measurements have also been made with dense aqueous gels of DNA in the absence of poly(ethylene oxide) at ordinary salt concentrations and in 6·0 M-LiCl. All preparations exhibit a well-defined interhelical spacing, implying substantial parallelism, but no lattice spacings higher than first order are observed. Comparison with calculated scattering curves for disoriented helical segments indicates that a structure very close to the B fiber structure prevails in all preparations. No significant contribution from the A or C fiber structures can be detected in either the condensed preparations or the LiCl solutions. Thus there is no basis for attributing the origin of the strongly anomalous circular dichroism spectra to a secondary structure significantly different from that in dilute solution. The decrease in interhelix spacing with increasing polymer concentration is in reasonable accord with expectation on the basis of excluded volume interactions. There is improved short-range order in the polymer-induced compact state as compared with the simple solution having the same interhelix spacing. The results are in reasonably good agreement with expectation for a folded chain structure of the compact state, similar to the usual mode of crystallization of simple linear polymers. There is no evidence for supercoiling.

DNA packing in single crystals inferred from freeze-fracture-etch replicas

Freeze-fracture-etch replicas of individual crystals of low molecular weight DNA grown from aqueous alcohol solution have been examined in the electron microscope. DNA crystals, like single, solution-grown crystals of other polymers, are thin, broad hexagonal plates with the DnA helix axes nearly perpendicular to the broad faces. Hexagonal spiral patterns are frequently seen on the faces, indicating the initiation of growth at a spiral dislocation. Since the distribution of crystal thicknesses closely corresponds to the distribution of contour lengths of the DNA preparation, it is inferred that the DNA molecules are fully extended rather than folded in the crystals. Uniformity of thickness within each crystal implies selection according to molecular weight during crystallization. High magnification examination of properly oriented fractures, together with digital averaging of two-dimensional densitometry of the electron micrographs, permits quantitative interpretation of the width and spacing of shadows cast by the edges of crystal planes. The shadows can be identified with planes consisting of single layers of helices. The intervals between shadows correspond to hexagonal packing, and the inter-helix spacing inferred from the electron micrograph measurements agrees well with an independent determination by X-ray diffraction at a similar temperature.

ELECTRON PARAMAGNETIC RESONANCE IN SINGLE CRYSTALS OF CUPRIC INSULIN.

THE binding of divalent metal ions to insulin has been the subject of several investigations, both chemical and physical in nature1–6. In describing here the application of electron paramagnetic resonance (EPR) spectroscopy to this problem, we wish to emphasize the richness of the data obtainable from orientation investigations of single crystals. This information is of the following kinds: (1) the number of distinct magnetic centres; (2) the orientation with respect to the crystal axes of the symmetry axes of the magnetic centres; (3) the symmetries of the protein environments of the magnetic centres, and the ground-states of the transition metal ions; (4) the delocalization of the electrons in metal ion–protein bonds; (5) identification of protein atoms bound to metal ions when the former have nuclear magnetic moments; (6) an estimate of the distance between metal ions.

Water of co-ordination in insulin†

Single-crystal electron paramagnetic resonance orientation studies, hydrogenion titration, and X-ray diffraction data have established that, in the rhombohedral insulin crystals which form at neutral pH, the two metal ions in a unit cell are located on a trigonal axis, each co-ordinated to three imidazole nitrogens. We have now obtained electron paramagnetic resonance spectra of deuterium oxide-substituted cupric insulin crystals. (The magnetic moment of the deuteron is about one-third that of the proton.) Resonance line narrowing is observed which, together with previous data, indicates that water molecules occupy the remaining co-ordination positions about each metal ion. We have used the measured decrease in line-width to calculate the average proton-to-metal distance. The copper to water-oxygen bond length was found to be in the range of corresponding values in the structures of small complexes determined by X-ray and neutron diffraction. This calculation of interatomic separation shows that it is essential to take into account the effect on the radial factor of the unpaired cupric spin delocalization onto the water oxygen. This delocalization was quantitated from the ligand hyperfine splitting arising from the co-ordinated nitrogens.