DONOR-DONOR ENERGY MIGRATION FOR DETERMINING INTRAMOLECULAR DISTANCESIN PROTEINS - I - APPLICATION OF A MODEL TO THE LATENT PLASMINOGEN-ACTIVATOR INHIBITOR-1 (PAI-1)

A new fluorescence spectroscopic method is presented for determining i ntramolecular and intermolecular distances in proteins and protein com plexes, respectively. The method circumvents the general problem of ac hieving specific labeling with two different chromophoric molecules, a s needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cystein e residues can be constructed for specific labeling with one or two id entical fluorescent probes, so-called donors (d). Fluorescence depolar ization experiments on double-labeled Cys mutant monitor both reorient ational motions oi the d molecules, as well as the rate of intramolecu lar energy migration. In this report a model that accounts for these c ontributions to the fluorescence anisotropy is presented and experimen tally tested. Mutants of a protease inhibitor, plasminogen activator i nhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata -3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migrat ion, the intramolecular distance between the d groups was calculated b y using the Forster mechanism and by accounting for the influence of l ocal anisotropic orientation of the d molecules. The calculated intram olecular distances were compared with those obtained from the crystal structure of PAI-I in its latent form. To test the stability of parame ters extracted from experiments, synthetic data were generated and rea nalyzed.