Investigation of GlpG Rhomboid Protease Using Site-specific 19F NMR

Abstract

The rhomboid family of intramembrane serine proteases are capable of cleaving transmembrane protein substrates embedded within the phospholipid membrane. This allows them to play a critical role in a wide range of biological processes enabling their use as potential targets for the development of therapeutic compounds. Although X-ray crystallography and functional studies have revealed insights into the rhomboid mechanism of proteolysis, questions remain regarding how transmembrane substrates gain access to the water-filled active site from the hydrophobic lipid environment. Currently, there exists a growing body of evidence suggesting that helix 5 (𝛼5) undergoes conformational dynamics that facilitate substrate entry into the rhomboid active site. In this thesis, 1D 19F solution-state NMR spectroscopy was used to monitor dynamics around the 𝛼5 gate of the rhomboid protease from E. coli, ecGlpG. This was achieved using site-specific labelling of cysteine residues with thiol-reactive probes bearing trifluoromethyl groups. A site in the interface of the 𝛼5 gate was labeled and well resolved 19F NMR spectra were obtained, showing evidence of two-state exchange. Although promising, complications from background labeling of a single endogenous cysteine residue required the generation of a cysteine knockout mutant. The cysteine knockout retained function and stability, but at lower levels than seen for the wild-type protein. Overall, these results demonstrate the potential for 19F NMR to be used to monitor 𝛼5 gating dynamics, although further work is needed to optimize labels and mutation sites.