title: Characterization of molten globule PopB in absence and presence of its chaperone PcrH.
abstract: The TTSS encoding "translocator operon" of Pseudomonas aeruginosa consists of a major translocator protein PopB, minor translocator protein PopD and their cognate chaperone PcrH. Far-UV CD spectra and secondary structure prediction servers predict an α-helical model for PopB, PcrH and PopB-PcrH complex. PopB itself forms a single species of higher order oligomer (15 mer) as seen from AUC, but in complex with PcrH, both monomeric (1:1) and oligomeric form exist. PopB has large solvent-exposed hydrophobic patches and exists as an unordered molten globule in its native state, but on forming complex with PcrH it gets transformed into an ordered molten globule. Tryptophan fluorescence spectrum indicates that PopB interacts with the first TPR region of dimeric PcrH to form a stable PopB-PcrH complex that has a partial rigid structure with a large hydrodynamic radius and few tertiary contacts. The pH-dependent studies of PopB, PcrH and complex by ANS fluorescence, urea induced unfolding and thermal denaturation experiments prove that PcrH not only provides structural support to the ordered molten globule PopB in complex but also undergoes conformational change to assist PopB to pass through the needle complex of TTSS and form pores in the host cell membrane. ITC experiments show a strong affinity (K(d) ~ 0.37 μM) of PopB for PcrH at pH 7.8, which reduces to ~0.68 μM at pH 5.8. PcrH also loses its rigid tertiary structure at pH 5 and attains a molten globule conformation. This indicates that the decrease in pH releases PopB molecules and thus triggers the TTSS activation mechanism for the formation of a functional translocon.
authors.affiliation: Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology (IICB), 4 Raja S.C. Mullick Road, Kolkata, 700032, India.
authors.forename: Supratim
authors.initials: S
authors.lastname: Dey
authors.forename: Abhishek
authors.initials: A
authors.lastname: Basu
authors.forename: Saumen
authors.initials: S
authors.lastname: Datta
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D005453:Fluorescence; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D025941:Protein Interaction Mapping; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014508:Urea / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D014364:Tryptophan; D014508:Urea