The formation of A-DNA in NaDNA films is suppressed by netropsin.

Oriented films of NaDNA complexed with netropsin were studied with deuterium nuclear magnetic resonance (2H NMR), X-ray diffraction and ultraviolet (UV) linear dichroism to obtain information about the influence of netropsin on the structural arrangement of the DNA bases and on the B-A transition. The results of these studies clearly demonstrate a strong suppression of the formation of A-DNA at relative humidities (RHs) down to about 50%. The suppression was complete in the NaDNA-netropsin complex studied with 2H NMR which had a netropsin input ratio, r, of 0.22 drug/base pair. The sample used for UV linear dichroism had a similar input ratio while the X-ray diffraction samples had input ratios between 0.033 and 0.39 drug/base pair. Together, the results of these studies are in agreement with previous infrared (IR) linear dichroism studies of the conformation of the sugar-phosphate backbone in NaDNA-netropsin complexes, which showed that the B-A transition is suppressed for r-values down to approximately 0.1 drug/base pair (Fritzsche, H., Rupprecht, A. and Richter, M., Nucleic Acids Res. 12 (1984) 9165-9177).     

Conformations of A-DNA and B-DNA in agreement with fiber X-ray and infrared dichroism

Two right handed double helices are proposed as models for DNA fibers in respectively the A and the B form. The present conformations have geometrical parameters which agree very well with fiber X-ray and the orientation of their phosphate groups is in good accordance with infrared dichroïsm measurements. Dihedral angles and complete sets of atomic coordinates are given together with stereo-views and curves of the calculated diffracted intensities. Results are compared with experimental data and with preceding DNA models.     

Stereochemical model for proflavin intercalation in A-DNA.

Linked-atom molecular modelling was employed to determine the steric and torsional requirements for intercalation of proflavine into a double-stranded region of DNA compatible with adjacent regions of cohelical A-DNA. The optimum intercalation conformation is characterized by the dihedral angles xi and psi becoming trans, with all sugars retaining the characteristics C3'-endo pucker. This extended conformation results in virtually no helical unwinding, suggesting it may be an appropriate model for an intercalative intermediary in mutagenesis by virtue of its similarity to standard helical DNA.     

A 2H-NMR study of the A-DNA conformation in films of oriented Na-DNA: evidence of a disordered B-DNA contribution

Solid-state ²H-nmr spectra have been obtained from folded films of oriented Li- and Na-DNA molecules with the purine bases selectively deuterium labeled at the 8 position. From line shape simulations, we find that the Na-DNA sample at 75% relative humidity (rh) contains both A-DNA and surprisingly large amounts of B-DNA

1 Here, B-DNA refers to “B-DNA family” (i.e. B- or C-DNA).

(57%). For the A-DNA component the average base tilt is 23°, and the total distribution width of tilt angles and helix axis orientations is ～ 4° (standard deviation). In the B-DNA component the base tilt is ～ 0° and the total distribution width is ～ 20°. In contrast, films of Li-DNA only exhibit the B-form line shape, consistent with a base tilt of ～ 0° and a total distribution width of base tilt angles and helix axis orientations of 9°. The nmr results that demonstrate the presence of large amounts of B-DNA in the Na-DNA sample contrast with the x-ray diffraction measurements that indicated mainly A-form. The nmr spectra are used to monitor the B-DNA content in the Na-films and to evaluate procedures for increasing the A-DNA fraction.     

A new infrared spectroscopoic marker for cochleate phases in phosphatidylserine-containing model membranes.

Fourier transform-infrared (IR) spectroscopic and electron microscopic studies are reported for 1,2-dimyristoylphosphatidylserine (DMPS) and for DMPS/1,2-dimyristoylphosphatidylcholine mixtures in the presence and absence of Ca2+ ion. The frequency of the methyl symmetric deformation mode near 1,378 cm-1, previously assumed insensitive to changes in lipid morphology, has been found to respond to cochleate phase formation by undergoing an approximately 8 cm-1 increase. The new IR spectroscopic marker at 1,386 cm-1 has been used to identify and verify structures suggested from the phase diagram of J. R. Silvius and J. Gagné (1984. Biochemistry. 23:3241-3247) for this system. In addition, the ability of Mg2+ ion to induce cochleate formation has been demonstrated. Higher Mg2+ than Ca2+ levels are required for this process. Finally, IR spectroscopy has been used to monitor dehydration of the lipid surface through changes in the asymmetric PO2- stretching mode. Dehydration precedes cochleate phase formation (i.e., occurs at a lower Ca2+/phosphatidylserine level).     

Displacements of backbone vibrational modes of A-DNA and B-DNA.

We display the displacement vectors or eigenvectors of calculations of the A- and B-DNA backbones. These calculations are based on a refinement scheme that simultaneously fit several backbone modes of A-DNA, B-DNA, and A-RNA. We discuss the role of symmetry operations in mode calculations and the relevance of these displacement vectors to the interpretation of linear dichroism measurements performed on the A- and B-DNA helix.     

The molecular electrostatic potential and steric accessibility of A-DNA.

The molecular electrostatic potential and steric accessibility of A-DNA are computed for base sequences (dG.dC)n and (dA.dT)n. An interpretation of the results in terms of the structure of A-DNA is provided and differences with respect to other forms of DNA, namely B-DNA and Z-DNA, are discussed.     

Ordered water structure in an A-DNA octamer at 1.7 A resolution

The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.     

A need for re-examination of the fibre diffraction data of A-DNA

A-DNA pattern, obtained using a flat plat camera, was indexed by Fuller $\text{et}\text{al}{}^6$ on the basis of a c-face centred monoclinic cell with a = 22.24 Å, b = 40.62 Å, c = 28.15 Å and β = 97.0°. A precession photograph of A-DNA which gives an undistorted picture of the lattice, showed that the unit cell parameters as given by Fuller $\text{et}\text{al}$ were not quite correct. The precession photograph showed a strong meridional reflection (R = 0.00 Å^?1) on the 11th layer line. But the occurrence of the meridional reflection on the 11th layer line could not be explained on the basis of the cell parameters given by Fuller $\text{et}\text{al}{}^6$; using those cell parameters the reflection which comes closest to the meridian on 11th layer line is at R = 0.025 Å^?1. However, a simple interchange of a and b values accounted for the meridional reflection on 11th layer line. The corrected cell parameter refined against 28 strong spots are a = 40.75 Å, b = 22.07 Å, c = 28.16 Å and β = 97.5°. In the new unit cell of A-DNA, the packing arrangement of the two molecules is different from that in the old one. Nonetheless, our earlier contention is again reaffirmed that both right and left-handed A-DNA are stereochemically allowed and consistent with the observed fibre pattern.     

Picosecond study of the near infrared absorption band of hemoglobin after photolysis of carbonmonoxyhemoglobin.

Picosecond absorption spectroscopy is used to examine the position and band shape of the near infrared absorption band of hemoglobin as a function of time after the photodissociation of CO from carbonmonoxyhemoglobin. For the earliest delay time probed, 35 ps, the peak of the transient spectrum is at 765 nm, red shifted by 6 nm from that characteristic of equilibrium deoxyhemoglobin. No evolution in either the peak position or band shape is observed for time delays up to 60 ns. In addition, the position and shape of the spectrum are independent of photolysis energies ranging from 15 microJ/pulse to 150 microJ/pulse, spanning conditions under which the photon/heme ratio is varied from 0.01 to 2.0. This indicates that the geometry in the heme group is unrelaxed and that equilibration of the surrounding protein structure occurs on a time scale longer than 60 ns.     

A new hereditary single band variant of the Gc system

Summary A new single band variant (Gc Ar) or the Gc subtypes not identical with the known Gc variants has been detected in the plasma of a healthy blood donor by isoelectric focusing. Using this technique the variant is represented by a single band which has a similar isoelectric point to the Gc 1C2 anodal band. It is well known that the single band Gc phenotypes remain unaltered after neuraminidase treatment. Nevertheless, the new single band variant (Gc Ar) is altered after neuraminidase treatment as is Gc 2A3. After neuraminidase treatment, the Gc Ar band is affected and moved to the nearby position of the Gc 2 band. Investigation of the proband's family shows that the variant occurs combined with the common alleles Gc 1F, Gc 1S and that it has an autosomal dominant inheritance.     

A note on crystal packing and global helix structure in short A-DNA duplexes

A simple relation exists between the packing density in crystals of short A-DNA duplexes and their global double-helical structure. The volume per nucleotide pair shows a linear inverse correlation with the mean displacement of base pairs from the best straight helix axis. The mean displacement is a measure of major groove depth and varies between -3.3 A and -4.9 A in A-form oligonucleotides analysed in the crystalline state. Since the mean displacement of base pairs from the helix axis determines other helical parameters such as base-pair longitudinal slide, its correlation with crystal packing is of considerable interest. The displacement-packing correlation is very clear for octamer duplexes which crystallize in three different lattices. Longer A-helical fragments sometimes deviate from the rule. It may be speculated whether A-form duplexes not completing a full helical turn are especially prone to distortions due to packing in crystals or arising from intermolecular contacts in solution.     

A re-examination of the crystal structure of A-DNA using fiber diffraction data

Classical A-DNA helices with h = 0.25 nm may represent the greatest mass per unit length attainable by polynucleotide duplexes. The X-ray diffraction pattern from polycrystalline and well-oriented fibers of calf thymus DNA in its A-form has been carefully re-examined. Indexing on the basis of a C-face-centered monoclinic unit cell of dimensions a = 2.170 nm, b = 3.990 nm, c = 2.803 nm and beta = 96.82 degrees is superior to alternatives that have been proposed. Two right-handed. Watson-Crick base-paired, helical DNA chains with 2 X 11 nucleotides per 2.803 nm pitch, each carrying C3'-endo furanose rings, pass through the unit cell. The crystallography requires the two chains in the duplex to be antiparallel and conformationally identical but the 11 nucleotides in each pitch may be distinct. However, a secondary structure with a mononucleotide asymmetric unit provides as good an X-ray agreement as one with 11 distinct nucleotides. This relative lack of variability is quite different from what is observed in fibrous B-DNAs.     

A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax

Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer.     

Adsorption of the protein antigen myoglobin affects the binding of conformation-specific monoclonal antibodies.

Five monoclonal antibodies against sperm whale myoglobin have been used to investigate the physical state of the antigen adsorbed onto a polydimethylsiloxane surface. The binding of each antibody is sensitive to the antigen's conformation in solution while the locations of the antigenic sites on the myoglobin molecule for three of the antibodies have been determined (Berzofsky, J.A., G.K. Buckenmeyer, G. Hicks, F.R.N. Gurd, R.J. Feldmann, and J. Minna. 1982. J. Biol. Chem. 257:3189-3198). The binding of the fluorescein isothiocyanate-labeled IgG and Fab antibodies to previously adsorbed myoglobin has been observed using total internal reflection fluorescence. Three of the antibodies bind specifically to surface-adsorbed myoglobin with affinities at least 50% relative to myoglobin in solution whereas two of the antibodies show affinities for the surface-adsorbed myoglobin diminished by at least two orders of magnitude relative to myoglobin in solution. The specific loss of certain antigenic determinants on the adsorbed myoglobin, coupled with the retention of others, indicates a nonrandom adsorption of the myoglobin molecules.