An Application Of Bimolecular Fluorescence Complementation (Bifc) For The Detection And Analysis Of Protein Interactions Along The Escherichia Coli Twin Arginine Translocation (Tat) Pathway
The twin arginine translocation (Tat) pathway of Escherichia coli possesses an innate ability to translocate fully folded proteins across the bacterial inner membrane; however an in vivo method to directly monitor the protein interactions involved in this pathway did not exist. By using yellow fluorescent protein bimolecular fluorescence complementation (YFP-BiFC), protein-protein interactions can now be visualized in unprecedented clarity and at near real time rates along the entirety of the Tat pathway. Two interacting proteins previously identified and characterized in the Tat pathway, DmsA and DmsD were chosen for YFP-BiFC proof of concept studies. Protein fusion chimeras were created, whereby YFP was split into two fragments, Y1 and Y2, and then attached to the C-terminus of DmsA and DmsD, respectively. Upon coexpression of the two chimeric proteins in vivo, DmsA and DmsD interacted, YFP was reconstituted, and upon excitation resulted in the emission of a fluorescent signal. To demonstrate the utility of YFP-BiFC beyond DmsA and DmsD, we made protein chimeras targeting every part of the Tat pathway. With these chimeras, we were able to detect a fluorescent signal for interactions between substrate-chaperone, substrate-machinery, chaperone-machinery, and machinery-machinery interactions. From these interactions, a quantitative fluorescent signal was obtained, showing a dynamic range in signal intensity depending on the type of interaction being monitored. Additionally, in vivo localization of the protein chimeras could be determined by fluorescence microscopy. Furthermore, we expanded the applicability of YFP-BiFC in four ways; 1) we generated a DmsD library to isolate higher affinity DmsA binding variants by screening for an increase in YFP-BiFC signal, 2) we used the irreversible association of YFP-BiFC to purify the DmsA and DmsD complex for crystallography studies, 3) we used a family of de novo designed 3-helix bundle proteins to investigate the ability of the Tat pathway to interact with proteins of varying degrees of stability, and 4) we were able to obtain a FRET signal between the DmsA-DmsD YFP-BiFC complex and TatC-CFP. Overall, YFP-BiFC is a powerful tool for monitoring protein interactions in vivo and as a stabilizing force for in vitro protein analyses.
dissertation or thesis