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).