Linear Dichroism as a Tool for Characterizing Strand Separation in DNA

Linear dichroism (LD) in combination with studies of UV absorption and circular dichroism is suggested as a method for characterizing denaturation and reassociation phenomena in DNA. Due to its strong dependence on the chain stiffness, LD sensitively reflects partial strand separations. In reassociated DNA, LD provides an index of the amount of ordered structure while hyperchromicity gives a measure of the amount of base-pairs reformed

High-sensitivity linear dichroism as a tool for equilibrium analysis in biochemistry- stability constant of DNA-ethidiumbromide complex

A stoichiometrical application of a sensitive method for linear dichroism (LD) detection is suggested for biochemical purposes. The complex formation between a binding site on a polynucleotide and a ligand may be studied with high precision if the following conditions are fulfilled: (1) The polymer can be given a fixed degree of orientation. (2) The site has a specific orientation with respect to the orientation axis of the polymer (e.g., intercalation). (3) The ligand has an anisotropic optical absorption property.The method was applied to studying the complex between DNA and ethidiumbromide, which was detected by LD with precision of \ub1 0.5 7 10−7 M in a 4 7 10−4 M DNA solution, i.e., 0.1% occupation of the total site concentration can be detected. The complsxation could be explained by a single type of site (n = 0.14 \ub1 0.01 sites per nucleotide residue) and a stability constant K1 = (2.5 \ub1 1) 7 105 M−1 at 0.2 M ionic strength.From the specific LD an average angle 60\ub0 was concluded between the helix axis and the long axis of the ethidiumbromide molecule.This value formally contradicts the Watson-Crick model or the intercalation model but may be explained by extension and deformation effects on the xhain by the flow

Optical studies on complexes between DNA and pseudoisocyanine

Linear dichroism (LD) results when pseudoisocyanine = PIC (1, 1-diethyl-2,2\u27-cyanine iodide) binds to flow-oriented DNA. LD may be used to follow the complexation both stoichiometrically and structurally, since when specified to unit complex concentration LD provides a measure of the average orientation of the absorbing transition dipole.Two different types of complexes can be distinguished: I. One strong, ionic-strength insensitive complex with monomeric PIC with an orientation indicating intercalation. II. Several weaker complexes of electrostatic nature (only observed at

Binding of methyl green to deoxyribonucleic acid analyzed by linear dichroism

From linear dichroism data a distinct structure of the strongest complex between the methyl green (MG) dication and the B-form DNA is concluded. MG is not intercalated but binds (probably to hydrophobic surfaces in the major groove) with its x and z axes at an angle to the local helix axis of 48\ub0 and 90\ub0, respectively. At higher occupation density, exciton coupling between transition dipoles of adjacent molecules is visualized in the linear and circular dichroism spectra. The final spectra may be understood in terms of a right-handed (MG)N helix in the major groove of the double helix

Linear dichroism studies of binding site structures in solution: Complexes between DNA and basic arylmethane dyes

The interaction between B-form DNA and twelve cationic triaryl-methane dyes was studied with respect lo optical properties and stabilities, using linear dichroism (LD) and aqueous two-phase partition techniques. Monovalent dyes derived from crystal violet as a rule form a single strong complex (K1 ca 105 M−1; site density per nucleotide base n1 ca 0.1 at 0.1M ionic strength) in which the plane of the dye is at an angle of less than 50\ub0 to the local DNA helix axis. The complex with fuchsin is weaker (104M−1) but can be explained by a similar orientation. For some of the dyes (those with pseudo-C2v symmetry) XXXre angular orientations of two molecule-fixed axes can be obtained. For the divalent methyl green a second complex appears to be formed at low ionic strength. Methyl green (and to some extent 2-thiophene green and malachite green) show exciton splitting in the LD spectrum and circular dichroism assignable to exciton coupling between transition dipoles roughly parallel to the helical strands, indicating a dye-dye interaction. Tne optical data, supported by fitting experiments with space-filling models, suggests a general structure for the binding site. The dye is not intercalated but is bound to exposed hydrophobic regions in the major groove. The ligand is in part (the charged amino groups) in contact with the phosphoribose chain but its main surface lies against the hydrophobic base-pair stack. For a diphenylmethane dye, Michler\u27s hydrol blue, a perpendicular orientation was observed, possibly due to intercaiation

Interaction Between DNA and 8-Methoxypsoralen Studied by Linear Dichroism

High sensitivity linear dichroism on DNA oriented by flow is shown to provide conclusive information about the interaction with 8–methoxypsoralen (8–MOP) both in the dark and by irradiation. 8–MOP forms directly in the dark a weak complex with DNA with a structure consistent with the intercalation model of Lerman. When aqueous DNA-psoralen solutions are UV irradiated (30800 nm). a photoadduct is formed. By comparison with experiments in ice matrix, it is suggested that it is the primarily bound 8–MOP that is responsible for the final product. It is inferred that this product is the 41,5I-photoadduct of 8–MOP. The orientation of the photo adduct on DNA is not consistent with intercalation

Cutinase-peptide fusions in thermoseparating aqueus two-phase systems : Prediciton of partitioning and enhanced tag efficiency by detergent addition

It is of increasing importance to develop efficient purification methods for recombinant proteins where the number of steps can be minimised. The aim has been to establish a method for predicting the partitioning of the wild-type target protein in an aqueous two-phase system, and with this as basis, develop fusion tags and optimise the phase system for enhanced partitioning of the target protein. The surface of the lipolytic enzyme cutinase from Fusarium solani pisi was investigated with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP). The accessible surface areas for the different amino acid residues were used together with peptide partitioning data to calculate the partition coefficient for the protein. The separation system was composed of a thermoseparating random copolymer of ethylene oxide and propylene oxide, Breox PAG 50A 1000, as top phase forming polymer and a hydroxypropyl starch polymer, Reppal PES 200, as bottom phase polymer. The calculated partition coefficient for the wild-type protein (K=1.0) agreed reasonably well with the experimentally determined value (K=0.85). Genetic engineering was used to construct fusion proteins expressed in Saccharomyces cerevisiae based on cutinase and peptide tags containing tryptophan, to enhance the partitioning in aqueous two-phase systems. The partitioning of the cutinase constructs could qualitatively be predicted from peptide partitioning data, i.e. the trends in partitioning could be predicted. A spacer peptide introduced between protein and tag increased the partitioning of the protein towards the ethylene oxide-propylene oxide (EOPO) copolymer top phase. The aqueous two-phase system was modified by addition of detergent to increase the partitioning of the cutinase variants towards the EOPO copolymer phase. Triton and a series of C12En detergents selectively increased the partitioning of cutinase constructs with (WP)4-based tags up to 14 times compared to wild-type cutinase. The protein partition could almost quantitatively be predicted from the peptide partition data