PMC:2724173 / 5274-7322 JSONTXT

{"project":"2_test","denotations":[{"id":"18304577-15701523-62526544","span":{"begin":1541,"end":1542},"obj":"15701523"},{"id":"18304577-10094626-62526545","span":{"begin":1642,"end":1644},"obj":"10094626"}],"text":"A full-length construct of MxiC (residues 1–355, MxiCFL) was purified by nickel-affinity chromatography followed by size-exclusion chromatography and revealed that MxiCFL elutes at a volume less than that of a monomer, but greater than that of a dimer (Fig. 1a). This result, combined with dynamic light-scattering data that revealed a major species with a larger than expected hydrodynamic radius (Rh ∼ 3.8 nm), suggested that MxiCFL does not possess a globular structure and may possess an elongated fold and/or be partially disordered. In crystallization trials, MxiCFL initially yielded three different crystal forms (two distinct P21 and one P43212) that diffracted only to 3.5–3.9 Å resolution. Selenomethionine-labeled MxiCFL yielded crystals (P43212 with a different cell) that diffracted to 3.2 Å resolution and were used for phasing by the multiple-wavelength anomalous diffraction (MAD) method (Table 1). Preliminary model building into these maps revealed that the first 70–80 residues were poorly ordered and not visible in the electron density. In addition, we observed that, in solution, the N terminus was susceptible to proteolytic degradation (Fig. 1b). Limited proteolysis with subtilisin followed by N-terminal sequencing and mass spectrometry revealed several degradation products resulting from cleavage of the N terminus up to residue 64. This proteolytically sensitive region of MxiC is equivalent to the region of YopN (32–76) that was shown to bind its chaperone and was disordered in the absence of this chaperone.5 Furthermore, in vivo, the stable expression and efficient secretion of YopN requires its chaperone.18 The proteolytic susceptibility of the N-terminal region of MxiC suggests that chaperone binding may act to protect MxiC from degradation via a similar mechanism. To date, a chaperone for MxiC has not been identified. In order to improve the quality of MxiC crystals, a shortened construct encompassing residues 74–355 (MxiCNΔ73) was expressed, purified (Fig. 1a) and subjected to crystallization trials."}