Understanding novel reconstitution systems for native membrane protein biophysical characterisation

Membrane Proteins (MPs) constitute the majority of developed drug targets, and our ability to satisfactorily investigate membrane proteins is paramount to a variety of areas within science. This focus has put a strong emphasis on understanding how best to imitate the local environment of membrane proteins to maintain a native-like conformation of target MPs for biophysical analysis. In particular, mass spectrometry has started to be used to characterise membrane protein-lipid interactions. Most commonly used detergent based MP reconstitution methods have been shown to remove some of these crucial lipids, altering MP characteristics. Because of this, reconstitution systems have been developed to improve flexibility in membrane protein research. However, a lack of understanding as to how well each of them replicates the native environment of membrane proteins hinders progress. Here, novel reconstitution systems (C6-C2-50, C8-C0-50 and G1 OGD modular detergent) are compared against current systems (SMA, DDM and A8-35) to provide understanding on how well each replicates the native membrane environment and how well they apply to different membrane environments. A model membrane protein, Bacteriorhodopsin (HbR), was expressed in its native host (H. salinarium) and heterologously expressed in E. coli for comparison. After membrane isolation, HbR and E. coli bacteriorhodopsin (EbR) was purified using a variety of detergents and nanoparticles. These preparations were analysed using native mass spectrometry and mass photometry to identify retained lipids and oligomerisation states. Our tested reconstitution systems demonstrated a mixture of advantages and disadvantages described in this thesis, that will be valuable in structural biology. Exploiting these findings will help further understand current limitations in membrane proteins structural investigations.