title: Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.
abstract: Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Ruiqiong
authors.initials: R
authors.lastname: Guo
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Kristen
authors.initials: K
authors.lastname: Gaffney
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
authors.forename: Zhongyu
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Miyeon
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Suttipun
authors.initials: S
authors.lastname: Sungsuwan
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Xuefei
authors.initials: X
authors.lastname: Huang
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.|Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Heedeok
authors.initials: H
authors.lastname: Hong
mesh_terms: D001710:Biotin; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D013057:Spectrum Analysis
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008565:Membrane Proteins; D001710:Biotin; D010450:Endopeptidases