Green fluorescent protein rendered susceptible to proteolysis: Positions for protease-sensitive insertions

The green fluorescent protein (GFP) is highly resistant to proteolysis and remains uncleaved after prolonged incubation with trypsin or pronase despit e several putative tryptic and chymotryptic sites in exposed loops. We have rendered GFP sensitive to proteolysis by inserting five amino acids, IEGRS , in loops at position 157, 172, or 189. Excitation and emission maxima of the three insertion mutants were similar to those of wild type, but quantum yields of mutants Omega 172 and Omega 189 were lower, indicating increased freedom of the fluorophore. Trypsin cleaved the native (folded) form of ea ch mutant at a unique site defined by the insert. Pronase also yields simil ar digestion patterns in these variants, but further proteolysis was also o bserved, suggesting that the primary cleavage relaxes GFP structure and rev eals previously inaccessible sites. Fluorescence of Omega 189 changed littl e upon digestion with trypsin but decreased progressively by as much as 40% upon digestion with increasing amounts of pronase. Fluorescence of other v ariants was not affected significantly by the proteases, further confirming the remarkable stabilities of GFP variants. These constructs define a new conformation-sensitive site around residue 189 of GFP and show that GFP may be useful for design of protease-susceptible molecules for monitoring of s pecific proteolytic activities in vivo. (C) 2001 Academic Press.