green-fluorescent-protein-based-expression-screening-membrane

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in <em>Escherichia coli</em>

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Diamond Light Source

Recent research has highlighted the presence of Toll/Interleukin 1 receptor (TIR)-domain proteins (Tdps) in a range of bacteria, suggested to form interactions with the human adaptor protein MyD88 and inhibit intracellular signaling from Toll-like receptors (TLRs). A Tdp has been identified in Yersinia pestis (YpTdp), a highly pathogenic bacterium responsible for plague. Expression of a number of YpTIR constructs of differing lengths (YpTIR1, S130-A285; YpTIR2, I137-I273; YpTIR3, I137-246; YpTIR4, D107-S281) as fusions with an N-terminal GB1 tag (the B1 immunoglobulin domain of Streptococcal protein G) yielded high levels of soluble protein. Subsequent purification yielded 4-6 mg/L pure, folded protein. Thrombin cleavage allowed separation of the GB1 tag from YpTIR4 resulting in folded protein after cleavage. Nuclear magnetic resonance spectroscopy, size exclusion chromatography, SDS-PAGE analysis and static light scattering all indicate that the YpTIR forms dimers. Generation of a double Cys-less mutant resulted in an unstable protein containing mainly monomers indicating the importance of disulphide bonds in dimer formation. In addition, the YpTIR constructs have been shown to interact with the human adaptor protein MyD88 using 2D NMR and GST pull down. YpTIR is an excellent candidate for further study of the mechanism of action of pathogenic bacterial Tdps.

Yersinia pestis TIR-domain protein forms dimers that interact with the human adaptor protein MyD88.

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95 ± 0.89 μM peptide and a Vmax of 53.91 ± 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein.

Article	Year
Yersinia pestis TIR-domain protein forms dimers that interact with the human adaptor protein MyD88. Microbial pathogenesis\| PubMed ID: 21640812	2011
Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35). Journal of bacteriology\| PubMed ID: 24078610	2013

Matthew Jennions

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