Developing a Universal Steric Trapping Strategy for Studying Folding and Stability of Helical Membrane Proteins

Abstract

“Steric trapping” is a method that links binding of monovalent streptavidin (mSA) to unfolding of a biotinylated protein (MP). It allowed the measurements of high affinity protein–protein interactions and thermodynamic stability of polytopic helical MPs in a native environment, which had been difficult to achieve using more conventional methods. In the current steric trapping framework, a conformation-sensitive chromophore or an enzymatic activity in a target MP is required for monitoring mSA-induced unfolding. This target-specific approach is a limiting factor that hinders its application to various MP systems, i.e. MPs in a misfolded conformation, MPs with an assembly role, or MPs without convenient unfolding readout. To further advance the steric trapping strategy for more general application, we have developed novel tripartite probes possessing a thiol-reactive group, a biotin group and a fluorescent or paramagnetic group for sensitization of unfolding or binding of mSA. We applied the new strategy to investigating the stability and unfolding mechanism of an intramembrane protease GlpG. By combining FRET between fluorescently labeled GlpG and quencher-labeled mSA as a measure of mSA binding and the proteolytic activity of GlpG as a measure of unfolding, we proved the thermodynamic coupling between binding and unfolding, and determined the thermodynamic stability and unfolding rate of GlpG in a native micellar environment. While the stabilities were similar independent of the location of biotin pairs, the unfolding was 20 times faster when the biotin pair was placed near the proteolytic active site. This result suggests a subdomain-organization of the helical bundle architecture of GlpG. Steric trapping may serve as a useful tool for elucidating the local versus global flexibility of helical MPs.