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Nat Chem Biol. 2016 May;12(5):353-360. doi: 10.1038/nchembio.2048. Epub 2016 Mar 21.

Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.

Author information

1
Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
2
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
3
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
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Contributed equally

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.

PMID:
26999782
PMCID:
PMC4837050
DOI:
10.1038/nchembio.2048
[Indexed for MEDLINE]
Free PMC Article

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