A staphylococcal anti-sigma factor possesses a single-domain, carries different denaturant-sensitive regions and unfolds via two intermediates

Abstract

RsbW, an anti-sigma factor possessing kinase activity, is expressed by many Gram-positive bacteria including Staphylococcus aureus. To obtain clues about the domain structure and the folding-unfolding mechanism of RsbW, we have elaborately studied rRsbW, a recombinant S. aureus RsbW. Sequence analysis of the protein fragments, generated by the limited proteolysis of rRsbW, has proposed it to be a single-domain protein. The unfolding of rRsbW in the presence of GdnCl or urea was completely reversible in nature and occurred through the formation of at least two intermediates. The structure, shape, and the surface hydrophobicity of no intermediate completely matches with those of other intermediates or the native rRsbW. Interestingly, one of the intermediates, formed in the presence of less GdnCl concentrations, has a molten globule-like structure. Conversely, all of the intermediates, like native rRsbW, exist as dimers in aqueous solution. The putative molten globule and the urea-generated intermediates also have retained some kinase activity. Additionally, the putative ATP binding site/catalytic site of rRsbW shows higher denaturant sensitivity than the tentative dimerization region of this enzyme.

A staphylococcal cyclophilin carries a single domain and unfolds via the formation of an intermediate that preserves cyclosporin A binding activity

“…The basic protein methods, namely, protein estimation, Western blotting, SDS-PAGE, and staining of polyacrylamide gel, were performed as reported [42, 56–58]. The theoretical mass of monomeric rCyp was determined by analyzing its sequence with ProtParam (web.expasy.org), a computational tool.…”

“…To know whether rCyp carries any domain, limited proteolysis of this protein was separately executed by different proteolytic enzymes using standard methods [58, 59]. Briefly, a buffer B [42] solution carrying rCyp (10 μM) and an enzyme (0.025 - 0.1 μM) was incubated at ambient temperature.…”

“…All of the aliquots were boiled prior to their resolution by a Tris-glycine SDS-13.5% PAGE. After staining with Coomassie brilliant blue, the photograph of acrylamide gel was captured as stated [58].…”

“…To know about the different structural elements of rCyp and rCyp-CsA in buffer B [42], the ANS fluorescence (λ ex / λ em = 360 / 400-600 nm), intrinsic tryptophan (Trp) fluorescence (λ ex / λ em = 295 / 300-400 nm), near-UV circular dichroism (CD) (250-320 nm), and far-UV CD (200-260 nm) spectra of these proteins were recorded essentially as described before [42, 51, 58]. We used 25 μM protein for the near-UV CD spectroscopy and 10 μM protein for the far-UV CD or the fluorescence spectroscopy.…”

“…To study the unfolding pathway of rCyp and rCyp-CsA, these proteins (10 μM each) were exposed to varying concentrations (0-8 M) of urea for ~18 h at 4°C as stated [51, 58]. Protein aliquots were always treated with the freshly prepared urea solution.…”