Tryptophan properties in fluorescence and functional stability of plasminogen activator inhibitor 1.
Plasminogen activator inhibitor 1 harbors four tryptophan residues at positions 86, 139, 175, and 262. To investigate the contribution of each tryptophan residue to the total fluorescence and to reveal the mutual interactions of the tryptophan residues and interactions with the other amino acids, 15 mutants in which tryptophan residues have been replaced by phenylalanines were constructed, purified, and characterized. Conformational distribution analysis revealed that the tryptophan mutants have a similar conformational distribution pattern as wild-type plasminogen activator inhibitor 1. Mutants in which tryptophan residue 175 was replaced by a phenylalanine displayed an increased functional half-life of the active conformation, whereas the functional half-life of mutants in which tryptophan residue 262 was replaced by a phenylalanine was substantially decreased. Comparative analysis of the fluorescence lifetimes, the extinction coefficients, and the quantum yields of the individual tryptophan residues demonstrates that tryptophan residue 262 gives the highest contribution to the total fluorescence. The other tryptophan residues have a very low quantum yield. In the wild-type protein, the fluorescence of all tryptophan residues is partially quenched as compared to the mutants that contain single tryptophan residues, due to conformational effects. The fluorescence of tryptophan residue 262 is very likely also partially quenched by energy transfer to tryptophan residue 175.
Verheyden
Stefan
S
Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, B3001 Leuven, Belgium.
Sillen
Alain
A
Gils
Ann
A
Declerck
Paul J
PJ
Engelborghs
Yves
Y
D007700:Kinetics; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D014364:Tryptophan