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Cyclophilin (Cyp), a peptidyl-prolyl cis-trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus. The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S. aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. Both the secondary structure and the tertiary structure of each intermediate appears very similar to those of the corresponding native protein. Conversely, the hydrophobic surface areas of the intermediates are comparatively less. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in the future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Triton X-100 (TX-100), a useful non-ionic surfactant, reduced the methicillin resistance in Staphylococcus aureus significantly. Many S. aureus proteins were expressed in the presence of TX-100. SarA, one of the TX-100-induced proteins, acts as a global virulence regulator in S. aureus. To understand the effects of TX-100 on the structure, and function of SarA, a recombinant S. aureus SarA (rSarA) and its derivative (C9W) have been investigated in the presence of varying concentrations of this surfactant using various probes. Our data have revealed that both rSarA and C9W bind to the cognate DNA with nearly similar affinity in the absence of TX-100. Interestingly, their DNA binding activities have been significantly increased in the presence of pre-micellar concentration of TX-100. The increase of TX-100 concentrations to micellar or post-micellar concentration did not greatly enhance their activities further. TX-100 molecules have altered the secondary and tertiary structures of both proteins to some extents. Size of the rSarA-TX-100 complex appears to be intermediate to those of rSarA and TX-100. Additional analyses show a relatively moderate interaction between C9W and TX-100. Binding of TX-100 to C9W has, however, occurred by a cooperative pathway particularly at micellar and higher concentrations of this surfactant. Taken together, TX-100-induced structural alteration of rSarA and C9W might be responsible for their increased DNA binding activity. As TX-100 has stabilized the somewhat weaker SarA-DNA complex effectively, it could be used to study its structure in the future. [ABSTRACT FROM AUTHOR]

SarA, a Staphylococcus aureus-specific dimeric protein, modulates the expression of numerous proteins including various virulence factors. Interestingly, S. aureus synthesizes multiple SarA paralogs seemingly for optimizing the expression of its virulence factors. To understand the domain structure/flexibility and the folding/unfolding mechanism of the SarA protein family, we have studied a recombinant SarA (designated rSarA) using various in vitro probes. Limited proteolysis of rSarA and the subsequent analysis of the resulting protein fragments suggested it to be a single-domain protein with a long, flexible C-terminal end. rSarA was unfolded by different mechanisms in the presence of different chemical and physical denaturants. While urea-induced unfolding of rSarA occurred successively via the formation of a dimeric and a monomeric intermediate, GdnCl-induced unfolding of this protein proceeded through the production of two dimeric intermediates. The surface hydrophobicity and the structures of the intermediates were not identical and also differed significantly from those of native rSarA. Of the intermediates, the GdnCl-generated intermediates not only possessed a molten globule-like structure but also exhibited resistance to dissociation during their unfolding. Compared to the native rSarA, the intermediate that was originated at lower GdnCl concentration carried a compact shape, whereas, other intermediates owned a swelled shape. The chemical-induced unfolding, unlike thermal unfolding of rSarA, was completely reversible in nature. [ABSTRACT FROM AUTHOR]

FKBP22, an Escherichia coli-encoded PPIase (peptidyl-prolyl cis-trans isomerase) enzyme, shares substantial identity with the Mip-like pathogenic factors, caries two domains, exists as a dimer in solution and binds some immunosuppressive drugs (such as FK506 and rapamycin) using its C-terminal domain (CTD). To understand the effects of these drugs on the structure and stability of the Mip-like proteins, rFKBP22 (a chimeric FKBP22) and CTD+ (a CTD variant) have been studied in the presence and absence of rapamycin using different probes. We demonstrated that rapamycin binding causes minor structural alterations of rFKBP22 and CTD+. Both the proteins (equilibrated with rapamycin) were unfolded via the formation of intermediates in the presence of urea. Further study revealed that thermal unfolding of both rFKBP22 and rapamycin-saturated rFKBP22 occurred by a three-state mechanism with the synthesis of intermediates. Intermediate from the rapamycin-equilibrated rFKBP22 was formed at a comparatively higher temperature. All intermediates carried substantial extents of secondary and tertiary structures. Intermediate resulted from the thermal unfolding of rFKBP22 existed as the dimers in solution, carried an increased extent of hydrophobic surface and possessed relatively higher rapamycin binding activity. Despite the formation of intermediates, both the thermal and urea-induced unfolding reactions were reversible in nature. Unfolding studies also indicated the considerable stabilization of both proteins by rapamycin binding. The data suggest that rFKBP22 or CTD+ could be exploited to screen the rapamycin-like inhibitors in the future. [ABSTRACT FROM AUTHOR]

Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors. [ABSTRACT FROM AUTHOR]