Examining the Effects of Netropsin on the Curvature of DNA A-Tracts Using Electrophoresis

A-tracts are sequences of repeated adenine bases that, under the proper conditions, are capable of mediating DNA curvature. A-tracts occur naturally in the regulatory regions of many organisms, yet their biological functions are not fully understood. Orienting multiple A-tracts together constructively or destructively in a phase has the potential to create different shapes in the DNA helix axis. One means of detecting these molecular shape differences is from altered DNA mobilities measured using electrophoresis. The small molecule netropsin binds the minor groove of DNA, particularly at AT-rich sequences including A-tracts. Here, we systematically test the hypothesis that netropsin binding eliminates the curvature of A-tracts by measuring the electrophoretic mobilities of seven 98-base pair DNA samples containing different numbers and arrangements of centrally located A-tracts under varying conditions with netropsin. We find that netropsin binding eliminates the mobility difference between the DNA fragments with different A-tract arrangements in a concentration-dependent manner. This work provides evidence for the straightening of A-tracts upon netropsin binding and illustrates an artificial approach to re-sculpt DNA shape.

Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15

Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties.

Determining Stability Constants of Complexes Between Ester Betulin Derivatives and β-Cyclodextrin by Affinity Capillary Electrophoresis

Inclusion complexes of ester betulin derivatives (pentacyclic lupane triterpenoids) with β-cyclodextrin (β-CD) were studied by affinity capillary electrophoresis. Stability constants were calculated from dependencies of viscosity-corrected effective electrophoretic mobilities on β-CD concentration (0-10 mM) in background electrolytes using nonlinear regression fitting. Logarithms of stability constants at 25 °C for 1:1 and 1:2 β-CD complexes of betulin 3,28-diphthalate, betulin 3,28-disuccinate and betulin 3,28-disulfate (95 % confidence interval) are 4.25 (4.16-4.32) and 7.27 (6.73-7.50), 4.38 (4.26-4.48) and 7.58 (6.90-7.84), 4.04 (4.00-4.08) and 5.91 (4.60-6.20), respectively

Spherical-Shape Assumption for Protein–Aptamer Complexes Facilitates Prediction of their Electrophoretic Mobility

The pdf file describes the results of this study, which aims at developing a model to accurately predict electrophoretic mobilities of protein–aptamer complexes. The excel file contains source data for electropherograms (signal vs time) which were obtained in this study and used to determine the migration times and electrophoretic mobilities of proteins, aptamers, ssDNAs, protein–aptamer complexes, and protein–ssDNA complexes. Additional sheets in the excel file contain values of all migration times and mobilities obtained from the electropherograms.

Spherical-Shape Assumption for Protein–Aptamer Complexes Facilitates Prediction of their Electrophoretic Mobility

The pdf file describes the results of this study, which aims at developing a model to accurately predict electrophoretic mobilities of protein–aptamer complexes. The excel file contains source data for electropherograms (signal vs time) which were obtained in this study and used to determine the migration times and electrophoretic mobilities of proteins, aptamers, ssDNAs, protein–aptamer complexes, and protein–ssDNA complexes. Additional sheets in the excel file contain values of all migration times and mobilities obtained from the electropherograms.