Abstract
Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein's initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.
Highlights
This class of proteins shares common structural features that maintain their stability in the lipid bilayer
We sought to identify specific surface-exposed extracellular loops of ShuA that are important for hemoglobin binding
24 variants of ShuA carrying deletion or alanine substitutions in each of the surface-exposed extracellular loops were displayed on an M13 bacteriophage surface and evaluated for display levels and hemoglobin binding
Summary
This class of proteins shares common structural features that maintain their stability in the lipid bilayer. Hydrophobic residues face the lipid bilayer exterior, and polar residues line the interior of the b-barrel This transmembrane channel can allow passage of polar ligands through the membrane and into the cell.[6]. We probed the structure and function relationships of key residues of a protein in a major class of b-barrel membrane proteins, the TonB-dependent transporters (TBDT) This class of MPs is anchored at the bacterial cell surface, and actively transports nutrients through its transmembrane channel into the cell for survival and virulence.[7] TBDTs use a proton motive force to generate the energy required for transport of essential nutrients across the outer membrane. These transporters directly interact with the TonB protein in the TonB–ExbB–ExbD complex located in the inner membrane to transduce energy from the proton motive force.[8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
