Peltier multicell holder

Fluorescence measurements with the wild-type and variant MCAD enzymes (0.5 μg/μl) diluted in 20 mM HEPES buffer, pH 7.0 and 200 mM NaCl were performed on a Cary Eclipse fluorescence spectrophotometer equipped with a temperature-controlled Peltier multicell holder (Varian). Intrinsic FAD fluorescence and binding of the hydrophobic dye 8-anilino-1-naphtalenesulfonic acid (ANS, Sigma-Aldrich) to surface exposed hydrophobic groups was co-monitored (multiwavelength program) during heat-induced denaturation experiments in the temperature range from 25 °C to 65 °C at a heating rate of 1 °C/min. Changes in ANS fluorescence emission were detected at 450 nm (excitation at 395 nm, 5.0/10.0 nm slit widths), whereas intrinsic FAD emission was monitored simultaneously at 530 nm (excitation at 450 nm, 5.0/10.0 nm slit widths). We ensured that no fluorescence resonance energy transfer occurred at the wavelengths used. Transition midpoints (T_m1/2) were determined graphically with T_m1/2 being the temperature at which half denaturation occurred. Significances between wild-type and variant proteins were calculated by one-way ANOVA followed by a Dunnett’s post test (GraphPad Prism 5.0).

Right angle light scattering (RALS) experiments were performed on a Cary Eclipse fluorescence spectrophotometer equipped with a temperature-controlled Peltier multicell holder (Varian). Samples contained 0.08 μg/μl protein diluted in 20 mM HEPES buffer, pH 7.0 and 200 mM NaCl. The increase in turbidity was monitored in the temperature range from 25 °C to 65 °C at a heating rate of 1 °C/min (excitation at 330 nm, emission 335 nm; 5.0 nm slit widths). Turbidity curves were illustrated with GraphPad Prism 5.0.

RALS experiments were performed on a Cary Eclipse fluorescence spectrophotometer with a temperature-controlled Peltier multicell holder (Varian, Palo Alto, CA, USA). Samples contained 0.6 mg/mL protein diluted in 20 mM HEPES buffer, pH 7.0, and 200 mM NaCl. The increase in turbidity was monitored in the temperature range from 25 to 62 °C at a heating rate of 1 °C/min (excitation at 330 nm, emission at 335 nm; 5.0 nm slit widths). Results were normalized by defining the smallest and largest mean value in each dataset as 0 and 100%, respectively. Each dataset includes triplicate samples. Normalization, curve modeling, and transition midpoints (from biphasic fitting) were calculated using GraphPad, San Diego, CA, USA.