Tryptophan properties in fluorescence and functional stability of plasminogen activator inhibitor 1

Abstract

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of the tryptophan residues and interactions with the other amino acids, 15 mutants in which tryptophan residues have been replaced by phenylalanines were constructed, purified, and characterized.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.

FIGURE 1

(A) Active conformation of PAI-1 with tryptophan residues 86, 139, 175, and 262, respectively highlighted in yellow, green, red and blue. (B) The different tryptophan residues in PAI-1 with their mutual distances, κ² values, and possible fluorescence quenchers.

FIGURE 2

Representation of a phase measurement with the fit against their two-exponential function, the respective residuals, and autocorrelation functions of (A) PAI-1-W139-175-262F and (B) PAI-1-W86-139-175F.

FIGURE 3

Representation of a phase measurement of PAI-1-W86-139-262F fitted with (A) a three-exponential function and (B) a two-exponential function, the respective residuals, and autocorrelation functions.