Fig 5. Biophysical characterization of MsCI4 and mutants.

(A) Far-UV CD spectra of wild-type (WT) MsCI4 and mutants MsCI4^WW/FF and MsCI4^WWK/FFX, and (B) corresponding melting curves, monitored at 222 nm. (C) Thermal stability of MsCI4 and binding deficient MsCI4^YW/AA in presence of thalidomide, uridine, thymidine and cytidine, as determined in the thermal shift assay. Only thalidomide and uridine lead to a shift in melting temperature with (two-tail) p-values of 1.5e-3 and 4.9e-2 as determined in a two sample equal variance t-test. (D) Tryptophan fluorescence spectra of MsCI4^WW/FF in presence of uridine, thymidine and cytidine. The emission maximum is shifted to shorter wavelength only in presence of the binder uridine, which can be indicative for the folding of the binding site. Note also the pronounced quenching effect indicative for binding. (E) same as (C) for the MsCI4^WWK/FFX mutant. As for the WT, thalidomide and uridine lead to a thermal shift, with p-values of 4.0e-4 and 1.2e-3. (F) same as (D) for the MsCI4^WWK/FFX mutant. The presence of uridine has the same effect as on the WT.