Final Report Summary - SMTAT (Single-molecule imaging of twin-arginine transporter assembly)
The twin arginine translocation system present in the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts transports folded proteins across the membrane in the presence of proton motive force. The transport required an orchestrated assembly of three major proteins, TatA, TatB and TatC. Several copies of these proteins assemble together to form a receptor complex and the translocase to allow transport of substrates of varying sizes.
-The main aim of this project was to evaluate the stoichiometry of the individual proteins in the transport complex. This was achieved using single molecule fluorescence detection using TIRF microscopy. The proteins were labelled in vivo via fusion tag to eyfp, cells were attached to cover slip using polylysine that was functionalized with a chemical fluorophore at substoichiometric concentration (Atto 532). A region of interest containing the cells and the chemical fluorophore was carefully photobleached to allow stepwise photobleaching of the chemical fluorophore. The photobleaching traces for the two fluorophores were plotted and normalised against their molecular brightness. Further calculations allowed the stoichiometry of the Tat complex to be determined. This work is novel in its approach and will create new grounds for the determination of the stoichiometry of membrane proteins using fluorescence. A manuscript is in preparation for this work.
-The other objective of this project was to achieve an in vitro bilayer system with the reconstituted Tat proteins. To this end, eyfp labelled Tat A, Tat B and Tat C were incorporated in Droplet interface bilayer systems, and their lateral diffusion constants were studied. The proteins were observed to be freely diffusing in the bilayer system, indicating successful reconstitution. Furtherwork is underway to assess the functionality of the proteins in artificial bilayers.
- As a minor deviation from the proposed plans, the fellow worked with the collaborators in combining single molecule fluorescence studies with molecular biology and molecular simulations to study the interactions between the Tat proteins at the molecular level. It was observed that in the TatBC receptor complex the transmembrane helix of each TatB molecule lies between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Tat A was also found to associate with TatC at the polar cluster site. This work provided a structural model for assembly of the active Tat translocase and demonstrated the ability of the combination of cutting edge techniques such as single molecule fluorescence and co-evolution analysis to predict protein interfaces in multi-subunit complexes. This work has led to a publication.
-The main aim of this project was to evaluate the stoichiometry of the individual proteins in the transport complex. This was achieved using single molecule fluorescence detection using TIRF microscopy. The proteins were labelled in vivo via fusion tag to eyfp, cells were attached to cover slip using polylysine that was functionalized with a chemical fluorophore at substoichiometric concentration (Atto 532). A region of interest containing the cells and the chemical fluorophore was carefully photobleached to allow stepwise photobleaching of the chemical fluorophore. The photobleaching traces for the two fluorophores were plotted and normalised against their molecular brightness. Further calculations allowed the stoichiometry of the Tat complex to be determined. This work is novel in its approach and will create new grounds for the determination of the stoichiometry of membrane proteins using fluorescence. A manuscript is in preparation for this work.
-The other objective of this project was to achieve an in vitro bilayer system with the reconstituted Tat proteins. To this end, eyfp labelled Tat A, Tat B and Tat C were incorporated in Droplet interface bilayer systems, and their lateral diffusion constants were studied. The proteins were observed to be freely diffusing in the bilayer system, indicating successful reconstitution. Furtherwork is underway to assess the functionality of the proteins in artificial bilayers.
- As a minor deviation from the proposed plans, the fellow worked with the collaborators in combining single molecule fluorescence studies with molecular biology and molecular simulations to study the interactions between the Tat proteins at the molecular level. It was observed that in the TatBC receptor complex the transmembrane helix of each TatB molecule lies between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Tat A was also found to associate with TatC at the polar cluster site. This work provided a structural model for assembly of the active Tat translocase and demonstrated the ability of the combination of cutting edge techniques such as single molecule fluorescence and co-evolution analysis to predict protein interfaces in multi-subunit complexes. This work has led to a publication.