Proteinase K cleaves flexible linker regions, and leaves β-barrels intact. The linker was cleaved by addition of 2 µL enzyme (5 mg/ml stock in H2O) to 18 µl sensor solution (in 10 mM NaPi, 100 mM NaCl, pH 7.4). After 1 minute 2 µl PMSF (100 mM in isopropanol) was added. (a) Fluorescence emission spectrum after mCerulean excitation (420 nm), before and after Proteinase K treatment. The mCitrine emission is absent after linker cleavage. (b) Fluorescence emission spectrum upon mCitrine excitation (515 nm). The mCitrine concentration does not decrease, verifying that the fluorophores are intact. (c) Effect of added Ficoll on the mCitrine/mCerulean ratio. Without proteinase K treatment the ratio increases, whereas the cleaved sensor does not show a FRET effect, excluding aggregation phenomena. (d) SDS-PAGE (10% polyacrylamide) analysis of the cleavage reaction, showing single bands before and after cleavage. The band after cleavage is less sharp, possibly due to the different cleavage sites of proteinase K in the linker. In-gel fluorescence was recorded using excitation at 460 nm, filter Y515.