Spontaneous formation of helical structures from phospholipid-nucleoside conjugates.

Phospholipid-nucleoside conjugates containing two myristoyl groups and a nucleotidyl group, collectively designated as dimyristoyl-5'-phosphatidylnucleosides, were enzymatically synthesized and their self-organization, morphology, and physicochemical properties investigated. The dimyristoyl-5'-phosphatidylnucleosides spontaneously assembled to form various types of helical strands. Neutral and alkaline solutions of dimyristoyl-5'-phosphatidyladenosine (DMPA) produced multihelical strands. The multihelical strand consisted of several single helical strands of approximately 50 A in diameter and helical pitch approximately 100 A. DMPA produced cigar-like scrolls (tubular structures) in acidic solution, which consisted of many double-helical strands aligned parallel to each other. Diacyl-5'-phosphatidyladenosine with a shorter chain length as long as an alkyl group, dilauroyl-5'-phosphatidyladenosine (DLPA), didecanoyl-5'-phosphatidyladenosine (DDPA), and dioctanoyl-5'-phosphatidyladenosine (DOPA) formed extended tape structures having double-helical strands aligned parallel. Dimyristoyl-5'-phosphatidylcytidine (DMPC) produced network structures at an early stage, which were slowly transformed into multihelical strands. The multihelical strands contained some single-helical strands of approximately 55 A in diameter and helical pitch approximately 150 A. DMPA produced no definite helical structure in acidic solution but rather large lamellar structures. Dimyristoyl-5'-phosphatidyluridine (DMPU) produced crystalline platelet structures of approximately 1000 A in width in both alkaline and acidic solution. A 1:1 mixture of DMPA and DMPU formed a new hybrid helical strand having a wide and thick ribbon structure of approximately 300 A in diameter and helical pitch approximately 2000 A. The formation of different helical strands and effects of chain lengths of alkyl groups and a nucleotidyl group in phospholipid-nucleoside conjugates on that of helical strands in aqueous solution are discussed.     

Light scattering measurements supporting helical structures for chromatin in solution.

Laser light scattering measurements have been made on a series of polynucleosomes containing from 50 to 150 nucleosomes. Radii of gyration have been determined as a function of polynucleosome length for different ionic strength solutions. The results suggest that at low ionic strength the chromatin adopts a loosely helical structure rather than a random coil. The helix becomes more regular on increasing the ionic strength, the dimension resembling those proposed by Finch and Klug for their solenoid model.     

Spontaneous formation of helical strands from nucleic acid-lipid conjugates

We have developed a new class of helical strands that self-assembled spontaneously in aqueous solution. Phospholipid-deoxynucleoside conjugates containing two myristoyl groups and a deoxynucleosidyl group spontaneously assembled to form different types of helical strands.     

Configurational changes in chromatids from helical to banded structures

Induction of configurational changes in the helical chromatids of air dried chromosomes was used to explore the mechanism of G-banding. From the water-Giemsa stained metaphase spreads of Chinese hamster cells, chromosomes having clearly helical chromatids were selected and photographed. Then the chromosomes were decolorized, treated with trypsin, and restained with saline-Giemsa (1 x SSC). Such procedures were repeatedly carried out upon the same chromosomes. Subsequent examination of the chromosomes showed that configurational changes from a helical structure to a banded structure had occurred. Some chromosomes revealed a variety of transitional changes between these two configurations. During the repeated G-banding treatments, the distances between bands along the same chromatids changed each time. The results obtained seem to indicate that the G-banding results from locally induced compaction of chromosomal materials along the chromatids.     

Formation and activity of covalent conjugates of poliovirus and ligands binding to cell surface structures

Disulfide-linked conjugates of poliovirus with streptavidin or concanavalin A were formed and the binding of the conjugates to mouse L cells that lack natural poliovirus receptors was studied. The conjugate with streptavidin was specifically bound to biotinylated L cells, but not to unmodified L cells. The conjugate with conA was bound to L cells in the absence of, but not in the presence of alpha-methyl mannoside. Incubation of L cells with bound conjugates did not produce virus, although the conjugates were highly infectious in HeLa cells, containing natural poliovirus receptors. This suggests that the artificially bound virus was unable to penetrate the L cells and start replication. The possibility that binding of the virus to the natural receptor is required for efficient infection is discussed.     

Averaging data derived from images of helical structures with different symmetries.

There are many examples of macromolecules that form helical tubes or crystals, which are useful for structure determination by electron microscopy and image processing. Helical crystals can be thought of as two-dimensional crystals that have been rolled into a cylinder such that two lattice points are superimposed. In many real cases, helical crystals of a particular macromolecule derive from an identical two-dimensional lattice but have different lattice points superimposed, thus producing different helical symmetries which cannot be simply averaged in Fourier-space. When confronted with this situation, one can select images corresponding to one of the observed symmetries at the expense of reducing the number of images that can be used for data collection and averaging, or one can calculate separate density maps from each symmetry, then align and average them together in real-space. Here, we present a third alternative, which is based on averaging of the Fourier-Bessel coefficients, gn,l(r), and which allows the inclusion of data from all symmetry groups derived from a common two-dimensional lattice. The method is straightforward and simple in practice and is shown, through a specific example with real data, to give results comparable to real-space averaging.     

The helical repeat of underwound DNA in solution.

Closed circular DNA was relaxed with a topoisomerase in the presence of varying concentrations of the intercalating dye, ethidium bromide, to create underwound, planar DNA rings. We directly determined the helical repeat of these DNA molecules by the Gaussian center method and found that it varied as a simple predicted function of the degree of underwinding and the helical repeat of relaxed, dye-free DNA. We discuss these results in light of a recent mathematical treatment of DNA structure which predicts that the helical repeat of supercoiled DNA molecules in solution obeys the same function.     

Formation of conjugates from ciprofloxacin and norfloxacin in cultures of Trichoderma viride

The formation of conjugates from two antibacterial fluoroquinolone drugs, ciprofloxacin and norfloxacin, was observed in cultures of Trichoderma viride that had been grown in sucrose-peptone broth and extracted 16 d after dosing with the drugs. Both conjugates were purified by high-performance liquid chromatography and found to be optically active. They were identified by mass and proton nuclear magnetic resonance spectra as 4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl ciprofloxacin and 4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl norfloxacin. The transformation of veterinary fluoroquinolones in the presence of fungi may have ecological significance.     

RNA double helices generated from crystal structures of double helical dinucleoside phosphates.

The dinucleoside phosphates ApU and GpC form right-handed anti-parallel double helical fragments within their crystal lattices. Using a least squares procedure, we have generated the extended double helices which these fragments represent. ApU corresponds to a double helix with 11.9 residues per turn and a pitch of 28. 1Å. The GpC double helix has 10.4 residues per turn and a pitch of 26. 9Å.     

Formation of 100 A filaments from purified glial fibrillary acidic protein in vitro.

Glial fibrillary acidic protein, which is specific to astroglia in the central nervous system, polymerizes in vitro into filaments similar to native ~ 100 Å filaments. Following purification from aqueous extracts of bovine brain by immunoaffinity chromatography, GFA ² protein is highly soluble in very low ionic strength solutions. Sedimentation equilibrium analysis of protein solutions in prefilament solvent conditions (2 mm-Tris · HCl, pH 7.8, 20 °C, containing 0.5 mm-dithiothreitol) indicates a paucidisperse mixture of species in solution with a typical range of apparent weight-average molecular weights from about 186,000 to 227,000. Between pH 6.0 and 8.0 the solubility is a function of pH and ionic strength as well as temperature, and precipitation is favored by lowering the pH or temperature and by raising the ionic strength. GFA protein associates in the form of filaments over a narrow range of pH and ionic strength; optimal conditions for polymerization of a 0.1 mg/ml protein solution are 100 mm-imidazole-HCl buffer (pH 6.8), at a temperature of 37 °C, and there is no requirement for co-factors. Filaments appear primarily as tangles of smooth curvilinear structures approximately 100 Å in diameter and of indefinite length, although some lateral association of filaments into thick bundles is also observed. While the formation of filaments is not affected by the presence or absence of reducing agent, under oxidizing conditions disulfide linkages form between protein subunits. Disassembly is achieved by dialysis against 2 mm-Tris · HCl buffer (pH 8.5), but this process is significantly enhanced by the addition of 0.5 mM-dithiothreitol during assembly and disassembly.These experiments clarify the role of GFA protein as the subunit of astroglialspecific intermediate filaments. In addition, they suggest that the 100 Å filament, as other components of the cytoskeleton, may assemble and disassemble in the glial cytoplasm.     

Characterization of low populated peptide helical structures in solution by means of NMR proton conformational shifts

A NOE independent NMR method is proposed to characterize unambiguously residues involved in low populated isolated peptide helices. The method is based on the comparison of amide and H alpha chemical shift changes originated upon the addition of stabilizing or denaturing agents with true helical conformational shifts that have been measured for the first time using an isolated model peptide helix, the one formed by Ac-(Leu-Lys-Lys-Leu)3-NHEt in aqueous solution.     

Triple helical polynucleotidic structures: an FTIR study of the C+ .G. Ctriplet.

Triple helixes containing one homopurine poly dG or poly rG strand and two homopyrimidine poly dC or poly rC strands have been prepared and studied by FTIR spectroscopy in H2O and D2O solutions. The spectra are discussed by comparison with those of the corresponding third strands (auto associated or not) and of double stranded poly dG.poly dC and poly rG.poly rC in the same concentration range and salt conditions. The triplex formation is characterized by the study of the base-base interactions reflected by changes in the spectral domain involving the in-plane double bond vibrations of the bases. Modifications of the initial duplex conformation (A family form for poly rG.poly rC, B family form for poly dG.poly dC) when the triplex is formed have been investigated. Two spectral domains (950-800 and 1450-1350 cm-1) containing absorption bands markers of the N and S type sugar geometries have been extensively studied. The spectra of the triplexes prepared starting with a double helix containing only riboses (poly rC+.poly rG.poly rC and poly dC+.poly rG.poly rC) as well as that of poly rC+.poly dG.poly dC present exclusively markers of the North type geometry of the sugars. On the contrary in the case of the poly dC+.poly dG.poly dC triplex both N and S type sugars are shown to coexist. The FTIR spectra allow us to propose that in this case the sugars of the purine (poly dG) strand adopt the S type geometry.     

Characterization at atomic resolution of peptide helical structures.

A survey of literature for the various types of helices experimentally observed in high-resolution single crystal x-ray diffraction analyses of peptides has allowed to determine accurate conformational and helical parameters for the various secondary structures such as the alpha-helix, the 3(10)-helix, the fully extended conformation (2(5)-helix) and the beta-bend ribbon spiral. For each of these structures the characteristic phi, psi conformational parameters, n, the number of residues per turn, h, the height per residues and p, the pitch of the helix are described.     

Higher-order structures of chromatin in solution.

Neutron scatter studies have been made on gently prepared chicken erythrocyte chromatin over a range of ionic strength. At low ionic strength the mass per unit length of the '10 nm nucleofilament corresponds to one nucleosome per 8--12 nm and a DNA packing ratio of between 6 and 9. From the contrast dependence of the cross-section radius of gyration of the nucleofilament the following parameters have been obtained; RgDNA' the cross-section radius of gyration (Rg) when DNA dominates the scatter; RgP, the cross-section Rg when protein dominates the scatter; Rc, the cross-section Rg at infinite contrast and alpha, the constant which describes the dependence of the cross-section Rg on contrast variation. From our understanding of the structure of the core particle, various arrangement of core particles in the nucleofilament have been tested. In models consistent with the above parameters the core particles are arranged edge-to-edge or with the faces of the core particles inclined to within 20 degrees to the axis of the nucleofilament. With increase of ionic strength the transition to the second-order chromatin structure has been followed. This gave the interesting result that above 20 microM NaCL or 0.4 mM MgCL2 the cross-section Rg increases abruptly to about 9 nm with a packing ratio of 0.2 nucleosome/mn and with further increase of ionic strength the Rg increases to 9.5 nm while the packing ratio increases threefold to 0.6 nucleosome/nm. This suggests a family of supercoils of nucleosomes which contract with increasing ionic strength. In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34-nm supercoil are discussed.     

Crystal and solution structures of an HslUV protease-chaperone complex

HslUV is a "prokaryotic proteasome" composed of the HslV protease and the HslU ATPase, a chaperone of the Clp/Hsp100 family. The 3.4 A crystal structure of an HslUV complex is presented here. Two hexameric ATP binding rings of HslU bind intimately to opposite sides of the HslV protease; the HslU "intermediate domains" extend outward from the complex. The solution structure of HslUV, derived from small angle X-ray scattering data under conditions where the complex is assembled and active, agrees with this crystallographic structure. When the complex forms, the carboxy-terminal helices of HslU distend and bind between subunits of HslV, and the apical helices of HslV shift substantially, transmitting a conformational change to the active site region of the protease.     

Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS

The filament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPgammaS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA::ATPgammaS::ssDNA were compared by small angle neutron scattering. A model was developed that allowed for an analytical solution for small angle scattering on a long helical filament, making it possible to obtain the helical pitch and the mean diameter of the protein filament from the scattering curves. The results suggest that the structure of the filaments formed by these two RecA proteins, and particularly their complexes with ATPgammaS, is conservative.