The capA gene (FTT_0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues. The gene is part of an apparent operon with genes encoding two other membrane proteins, CapB, and CapC. Together, these proteins have been proposed to be involved in the biosynthesis of a polymeric-based capsule in this bacterium1,2. Membrane topology and secondary structure predictions show that CapA may have two transmembrane helices located at the N- and C-termini and a very large hydrophilic domain which is predicted to be mostly α-helical. We have overexpressed the capA gene in E. coli as a His6-tagged fusion with a folding reporter green fluorescent protein (GFP). Dynamic light scattering indicated that the purified CapA-GFP was highly monodisperse with a size that was dependent upon the protein concentration and choice of detergent. Circular dichroism showed that CapA-GFP was stable over a wide range of pH values. The CapA-GFP protein requires a high ionic strength to keep its secondary structure, indicating that electrostatic interactions are playing a key role in the stability of the protein. Analysis of the sequence by disorder predictors reveals that the CapA membrane protein contains a long disordered region of approximately 60 residues, suggesting that the ionic strength dependence arises due to electrostatic interactions involving the disordered region. Specifically, the addition of salt might minimize unfavorable intra- or inter-molecular electrostatic repulsions in the disordered region, allowing the formation of α-helices. Proteolysis experiments using proteinase K also confirmed that this disordered region is highly solvent exposed, which may indicate that it may play a role in binding to other proteins.
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