Qs cuvettes

CD spectroscopic experiments were used to study the solution structure of analogues in phosphate buffer (10 mM, pH 7.4) and in 50% 2,2,2-trifluoroethanol (TFE) using a Jasco J-815 spectropolarimeter in Hellma QS cuvettes with pathlength 1.00 mm. Stock solutions of 1 mg/mL were prepared from lyophilized analogs in 50 mM Na₂HPO₄, pH 7.4 and were diluted to appropriate concentrations of ~0.1 mg/mL in 10 mM buffer, or same concentration in 50% TFE (v/v). The spectra of the analogues were recorded in 10 mM buffer and 50% (v/v) TFE at room temperature. The measurements were conducted in the far UV range from 250 to 190 nm with a scan rate of 20 nm/min at 1 nm bandwidth, and a time constant of 0.5 s. The resulting spectra were the average of six separate recordings. Blank samples of 10 mM buffer, respectively 10 mM buffer with 50% (v/v) TFE, were recorded and subtracted from the relevant sample spectra. The CD spectra were normalized with regard to the UV absorbance at 280 nm (relative to the D2D analog). The UV absorbance data were measured on a Shimadzu UV-3600 UV–vis–NIR spectrophotometer using a 10 mm Hellma quartz cuvette. Finally, the CD spectra were smoothed with a five-point Savitzky–Golay filter in Jasco spectra analysis and the CD signal at 250 nm was adjusted to zero.

Purified pyrene-labelled rabbit G-actin, (Cytoskeleton Inc.) at 0.6 pyrene/monomer equivalence, was prepared at 2.5 μM in 5 mM Tris–HCl pH 8.0, 0.2 mM CaCl₂. An equal volume of aldolase, CTD, or bovine serum albumin (up to 50 μM final concentration) or no protein was then added prior to polymerization. Actin polymerization was initiated by transfer at a 1 in 10 ratio to 500 mM KCl, 20 mM MgCl₂, 10 mM ATP according to the manufacturer's instructions, in 10 mm QS cuvettes (Hellma GmbH & Co. KG). Fluorescence measurements were performed in a JASCO FP-6300 spectrofluorimeter at 20 °C and the fluorescence emission intensity recorded for 10 min using an excitation wavelength of 366 nm and an emission wavelength of 407 nm as described previously for pyrene-labelled actin [33] (link). The rate of actin polymerization was measured as the increase in fluorescence intensity per min. All reactions were carried out in duplicate.