title: Reversible Unfolding of Rhomboid Intramembrane Proteases.
abstract: Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.
authors.affiliation: Department of Biochemistry, Membrane Protein Disease Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Panigrahi
authors.affiliation: Department of Biochemistry, Membrane Protein Disease Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Elena
authors.initials: E
authors.lastname: Arutyunova
authors.affiliation: Department of Biochemistry, Membrane Protein Disease Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Pankaj
authors.initials: P
authors.lastname: Panwar
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Gimpl
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany.
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
authors.affiliation: Department of Biochemistry, Membrane Protein Disease Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada. Electronic address: mlemieux@ualberta.ca.
authors.forename: M Joanne
authors.initials: MJ
authors.lastname: Lemieux
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006193:Haemophilus influenzae / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D055503:Protein Multimerization; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011532:Providencia / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; D010450:Endopeptidases