Purification and Biophysical Characterization of the CapA Membrane Protein FTT0807 from Francisella tularensis

Abstract

The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3–9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50–60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides.

Purification and structural characterization of Mce4A from Mycobacterium tuberculosis

“…Many proteins show that the disordered region can be converted into ordered conformation on the addition of neutral salts [35]. On the contrary, it is seen in Fig.…”

From the Outside-In: The Francisella tularensis Envelope and Virulence

“…Indeed, as noted earlier, Apicella et al, found that capB and capC were not involved in LVS capsule production, suggesting that the cap genes perform another function. A more recent study suggested that capA may encode an integral inner membrane protein that interacts with CapB and CapC to export carbohydrates extracellularly (Martin-Garcia et al, 2014 ). Given the large number of genes and operons that have been reported to play roles in F. tularensis capsule production, more studies are clearly needed.…”

Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation

“…Flpp3 has been identified as a virulence determinant (Su et al, 2007), and its atomic resolution structure has recently been determined by NMR (Zook et al, 2015); however, a crystallographic structure has not yet been reported. Proteinase K is a commercially available enzyme belonging to the family of serine proteases, which has been used in numerous studies to identify disordered regions in intrinsically disordered proteins (IDPs;Csizmó k et al, 2005;Denning et al, 2003;Marín et al, 2012;Martin-Garcia et al, 2014;Nyarko et al, 2004). Thaumatin and lysozyme were also included as they crystallize rapidly and easily, and are therefore frequently used as model systems in protein crystallization studies and many proof-of-principle studies.…”