1. Dissecting binding of a β-barrel membrane protein by phage display.
- Author
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Meneghini LM, Tripathi S, Woodworth MA, Majumdar S, Poulos TL, and Weiss GA
- Subjects
- Amino Acid Sequence, Bacterial Outer Membrane Proteins genetics, Bacterial Outer Membrane Proteins metabolism, Bacteriophage M13 metabolism, Binding Sites, Capsid Proteins genetics, Capsid Proteins metabolism, Cell Surface Display Techniques, Cloning, Molecular, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Heme metabolism, Hemoglobins chemistry, Hemoglobins metabolism, Histidine chemistry, Histidine metabolism, Models, Molecular, Protein Binding, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Structure, Tertiary, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Shigella dysenteriae metabolism, Bacterial Outer Membrane Proteins chemistry, Bacteriophage M13 genetics, Capsid Proteins chemistry, Heme chemistry, Recombinant Fusion Proteins chemistry, Shigella dysenteriae genetics
- 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.
- Published
- 2017
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