Chirascan plus qcd

Absorption measurements were recorded with a Lambda 35 spectrometer (PerkinElmer, Waltham, MA, USA) using 1 cm optical path quartz cuvettes. Fluorescence measurements were conducted with LS55B spectrofluorometer (PerkinElmer, Waltham, MA, USA) equipped with polarizers, thermostated cuvette compartments, and magnetic stirring. Induced circular dichroism spectra were registered by Chirascan-plus qCD (Applied Photophysics Limited, Surrey, UK), which was equipped with thermostat. All spectroscopic measurements were carried out at a room temperature (23–25 °C). Optical density of all samples did not exceed 0.4 a.u. All of the measurements were performed in triplicate.

Double stranded udpO (5′-ATTTATGCAACGCA-3′) tagged with ALEXA532 at 5′ end was purchased from IBA Lifesciences (the binding site is highlighted in bold). Experiments were performed in 50 mM sodium phosphate, 30 mM sodium chloride and 1 mM EDTA, pH 6.0 buffer. A starting DNA concentration of 300 nM was titrated with different concentrations of CytR ranging from ∼1 nm to 100 μM; the resulting change in anisotropies were monitored following a 5 minute equilibration at every titration step by exciting the dye at 530 nm and collecting the emission at 580 nm in a Chirascan Plus qCD instrument (Applied Photophysics Ltd., UK) equipped with a fluorescence polarization accessory.

Circular Dichroism (CD) experiments were performed in a Chirascan-plus qCD (Applied Photophysics Ltd, Leatherhead, Surrey, UK) in a 0.1 cm path length quartz cuvette. AbeTx1 and analogs (10 µM) were added in a cuvette containing CD Buffer or CD Buffer and POPC (100 mol%) or POPC: POPS (80:20 mol%) LUVs (100 µM). The CD spectra were collected from 190 to 260 nm, at 25 °C, with a bandwidth of 1 nm, step of 1 nm, time-per-point of 0.1 s, and current time-per-point of 0.1 s. Each CD spectra was averaged over eight repetitions. The observed ellipticity θ (mdeg) was converted to mean amino acid ellipticity θ (deg·cm²·dmol⁻¹) after the baseline correction, using the relationship: [θ] = (100.θ)/(l.c.n). Where l is the path length in centimeters, c is peptide millimolar concentration and n the number of peptide amino acids. A lipid baseline spectrum was subtracted from all peptide/lipid spectra.

The self-assembly of H-NS as a function of protein concentration was investigated by monitoring changes in fluorescence anisotropy specifically attributed to the tryptophan residue (W109) located in its C-terminal DNA binding domain. To study this, a tryptophan null variant of H-NS (W109F; termed H-NSm) was generated. The experiments were carried out in a 150 mM ionic strength buffer at 25 and 37°C. Here, a 2 μM wild-type H-NS solution was excited at 295 nm in the presence of varying concentrations of the H-NS tryptophan variant (H-NSm) ranging from 30 nM to 50 μM (final volume of 2.2 ml). Changes in anisotropy were monitored after a five-minute equilibration period at each temperature. Excitation of the H-NS sample was performed at 295 nm, and the emitted light at 340 nm was collected using a Chirascan-plus qCD instrument with a fluorescence polarization (FP) accessory equipped with a Peltier unit (Applied Photophysics, UK), and the data was acquired at every 20 s time per point. A cut-off filter of 320 nm was placed before the FP detector to filter the emitted photons.

N36 and AP3 or AP3P4E were dissolved in ddH₂O and PBS (1×, pH 7.4), respectively, at a final concentration of 10 μM. Equimolar mixtures were incubated at 37 °C for 30 min. CD spectra of each sample were recorded on a Chirascan Plus qCD (Applied Photophysics, Leatherhead, UK) using a 1 nm bandwidth with a 1 nm step resolution from 195 to 260 nm at room temperature. The spectra were corrected by subtracting a blank corresponding to the solvent composition of each sample. The CD data are shown as the mean residue ellipticity, and the mean residue ellipticity at 222 nm ([θ]_222nm) divided by the expected value of 100% α-helix formation (−33,000° cm²/dmol) was calculated to obtain the α-helical content. The thermal denaturation experiment was performed by applying a thermal gradient of 2 °C/min and monitoring the change in ellipticity from 20 °C to 90 °C at 222 nm.