General Steric Trapping Strategy Reveals an Intricate Cooperativity Network in the Intramembrane Protease GlpG under Native Conditions

Abstract

of monocytes. Association of LBP with distinct membrane domains is shown in analysis of DRM membrane fractions from primary monocytes. Studies on HEK293 cells and human macrophages demonstrate that LBP-binding to host cell membranes occurs independently of the TLR4-receptor and is involved in the uptake and intracellular transport of LPS. Conclusions: In addition to its function in serum, LBP is also abundant in the cytoplasmic membrane and in intracellular compartments of monocytes and macrophages. Cell interaction, localization and intracellular LPS transport by LBP occur independent of TLR4, suggesting a new role of LBP in intracellular LPS-trafficking and signalling. This work was funded in parts by Leibniz-Graduate School ''Model systems for infectious diseases'' and the Cluster of Excellence ''Inflammation at Interfaces'' project EXC306OTP4 to A.B.S. Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment has been a necessary but challenging. Here we present a set of methods for analyzing key elements in membrane protein folding, including thermodynamic stability, compactness of the unfolded state and unfolding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doublybiotinylated protein to 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 those methods to an 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 for maintaining the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins. 1278-Pos Board B255In Cell Footprinting Coupled with Mass Spectrometry for the Structural