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 despite 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 Omega172 and Omega189 were lower, indicating increased freedom of the fluorophore. Trypsin cleaved the native (folded) form of each mutant at a unique site defined by the insert. Pronase also yields similar digestion patterns in these variants, but further proteolysis was also observed, suggesting that the primary cleavage relaxes GFP structure and reveals previously inaccessible sites. Fluorescence of Omega189 changed little upon digestion with trypsin but decreased progressively by as much as 40% upon digestion with increasing amounts of pronase. Fluorescence of other variants 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 specific proteolytic activities in vivo.
Chiang
C F
CF
Department of Chemistry, Clark Atlanta University, Atlanta, Georgia 30314, USA.
Okou
D T
DT
Griffin
T B
TB
Verret
C R
CR
Williams
M N
MN
D001665:Binding Sites / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000737:chemistry*; D011402:Pronase / Q000737:chemistry; D017433:Protein Structure, Secondary / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000502:physiology; D014357:Trypsin / Q000737:chemistry
D008164:Luminescent Proteins; D010446:Peptide Fragments; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases; D014357:Trypsin; D011402:Pronase