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of the tryptophan residues and interactions with the other amino acids, 15 mutants in which tryptophan residues have been replaced by phenylalanines were constructed, purified, and characterized.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

Metals affect the structure and activity of human plasminogen activator inhibitor‐1. II. Binding affinity and conformational changes

“…Consistently, using a similar time window, mixing with varying concentrations of either of these metals resulted in no significant changes in the observed rate constant for either exponential, indicating that these changes are conformational events that occur after binding. Thus, in contrast with what is observed with the Type II metals, the binding event for Type I metals is not detected by perturbations in any of four tryptophan residues (86, 139, 175, or 262), which are the main contributors to PAI‐1 fluorescence using this method,44 so that the binding event is not reported directly. Instead, it appears that changes in the microenvironment of these residues are only observed subsequent to binding and are associated with conformational changes in PAI‐1.…”

Probing the folding pathway of a consensus serpin using single tryptophan mutants

“…The sum of the emission spectra of all single tryptophan variants yielded a maximal intensity about 120% of that of cAT, with comparable peak emission wavelengths at 331–332 nm. A previous analysis of plasminogen activator inhibitor-1, with three of the four tryptophan residues at identical positions to those in cAT, revealed significant quenching of Trp275 by Trp194 49 . In the context of similar inhibitory activity, far-UV CD profiles and the peak fluorescence emission wavelength across the three cAT variants, this 20% increase in intensity was therefore more likely the consequence of resonance energy transfer in cAT rather than the result of marked structural perturbation.…”

“…Tryptophan variants can be used as site-specific probes of the local folding behaviour of a protein, as described previously for α 1 -AT 47 , 48 and plasminogen activator inhibitor-1 49 . The cAT sequence contains tryptophans at positions 160, 194 and 275 (all designations made using α 1 -AT numbering), corresponding with helix F, the loop connecting strand 3 of α-helix A and strand 4 of β-sheet C (situated in the ‘breach’ region at the top of β sheet A), and helix H respectively (Figs 1A and 5A ).…”

Algorithm for the Analysis of Tryptophan Fluorescence Spectra and Their Correlation with Protein Structural Parameters

“…Classification of spectral and structural parameters of tryptophan residues in proteins within five discrete classes (presented above) are already accepted and widely used [78][79][80][81][82][83][84][85][86]. Here we would like to provide several examples of the application of our algorithms in the study of protein structure and dynamics.…”