UV spectroscopy
Second-derivative UV spectroscopy was used to detect conformational changes. Measurements were performed on an Agilent 8453 UV-Vis spectrometer (Agilent Technologies, Waldbronn, Germany). Samples were measured in 2 ml half-micro quartz cuvettes (Hellma Benelux, Kruibeke, Belgium) with a path length of 10 mm in a concentration of 1 mg/mL. Absorbance was measured from 248 to 332 nm with 1 nm intervals and an integration time of 15 seconds. Background correction was performed using solvent. Second-derivative spectra were calculated with UV-Visible ChemStation software (Agilent Technologies, Walbronn, Germany) as described earlier (20). The a/b ratio, i.e., the ratio between (a) the vertical distance between the peak minimum at 283 nm and the maximum at 287 nm and (b) the vertical distance between the minimum and maximum at 290 nm and 295 nm was calculated and used to determine the exposure of tyrosine residues to bulk solvent, which is sensitive to changes in the tertiary structure (21).