Single Molecule Studies of Plasminogen Activator Inhibitor

One of the unique advantages of studying single molecule kinetics is to distinguish intermediates of multiple reaction pathways because it eliminates ensemble averaging. It had been suggested that the interaction between proteinase and Plasminogen Activator Inhibitor-1 (PAI-1) involves different reaction pathways, resulting in either successful inhibition with stable covalent PAI-1-proteinase complex formed or unsuccessful inhibition with free proteinase released from the complex. By observing the interaction between single PAI-1 and trypsin molecules, we attempt to identify the intermediate steps that lead to the two different reaction pathways, which are not yet clearly understood with the results of ensemble studies. In our single molecule experiments the major conformational changes that involves in the interaction between PAI-1 and trysin are studied by Forster Resonance Energy Transfer (FRET) between a donor, Alexafluo-488, attached to PAI-1 and an acceptor, Alexafluo555, attached to trysin. A CCD camera is used to follow the fluorescence from single pairs of PAI-1-Alexafluo-488 and trysin-Alexafluo-555, which gives us information of FRET efficiency and therefore the reaction kinetics in the PAI-1 and trypsin interaction.

Figure. Fluorescence image and trajectories of single Plasminogen Activator Inhibitor-1 (PAI-1) molecules labeled with Alexfluo-488. The majority of labeled PAI-1 molecules exhibited fluorescence characteristics as trace a, indicating photobleaching of the single dye molecule covalently attached to PAI-1. A few single labeled PAI-1 molecules showed step-wise fluorescence loss as trace b. Although photobleaching of multiple dye molecules, probably the result of overlabeled PAI-1, could easily account for such behavior, possibility of other dynamic behavior of PAI-1 could not be excluded. Some molecules also showed dynamic blinking of fluorescence as trace c, indicating the existence of dynamic processes, probably conformational fluctuation of PAI-1, which resulted in distinctive quenching of the dye fluorescence. However, the mechanisms of these dynamic processes are not yet known to us.

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