Membrane proteins are a unique class of proteins which reside within cellular membranes. They comprise 20223C30% of all proteins in most organisms. Membrane proteins are involved in a variety of important cellular processes including ATP synthesis, photosynthesis, catalysis, molecular transport and cell signaling. Missense mutations in the genes encoding membrane proteins cause several life-threatening diseases including cystic fibrosis, Alzheimer's disease, and Charcot-Marie Tooth's disease. These mutations are known to cause disease majorly by impacting protein stability, rather than function, via two mechanisms: 1) protein destabilization which leads to excessive degradation and low accumulation of functional protein, 2) stabilization of non-functional misfolded forms of a protein which overwhelm cellular degradation machinery. To fundamentally understand disease mechanisms, it is necessary to understand the molecular forces and mechanisms in the folding of membrane proteins. Although the study of protein folding has been one of the major quests in molecular biology over the last 223C60 years, the understanding of membrane protein folding lags far behind that of soluble proteins. This is primarily due to the lack of available methods to control the reversible folding of membrane proteins under native conditions. Recently, steric trapping, which couples the unfolding of a doubly-biotinylated protein to monovalent streptavidin binding, has emerged as a promising technique to study membrane protein folding directly under native conditions without the use of chemical denaturants, heat, or pulling force. This work presents generalized steric trapping techniques utilizing novel tripartite chemical probes to dissect the folding energy landscape of the intramembrane protease GlpG from Escherichia coli. The new steric trap tools were employed to examine the thermodynamic stability of GlpG and the physical dimension of its unfolded state. Upon the discovery of subglobal unfolding events of GlpG in the region encompassing the active site, an intricate cooperativity network important for maintaining the stability of GlpG was identified using cooperativity profiling at side chain resolution. Finally, double-mutant cycle analysis coupled with stability measurement by steric trapping revealed the weakly coupled hydrogen bond network in the catalytic active site of GlpG.
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