Introducing spin labels into proteins to determine their solution conformation by pulsed EPR methods

Abstract

In bacteria, mechanosensitive ion channels are essential for the cellular response to hypoosomitic shock, relieving the build up of membrane pressure. This thesis focuses on the Mechanosensitive Channel of Small Conductance (MscS) for which three conflicting gating models exist. These models were generated from structural studies of the closed and open conformations of MscS using three different experimental techniques. Pulsed Electron Double Resonance (PELDOR) spectroscopy was applied to MscS in the detergent solubilised state and the membrane-like bilayer. The distances between selectively introduced unpaired electrons in MscS were measured. PELDOR data in the detergent solubilised state were only consistent with the crystal structure depicting an open conformation of MscS, indicating that the transmembrane helices were unperturbed during crystallisation in detergent. MscS was reconstituted into membrane bilayer mimics, bicelles and nanodiscs, and PELDOR data in these environments suggested that both closed and open conformations determined by X-ray crystallography are stabilised in the membrane bilayer. The second part of this thesis involved stabilisation of an enzyme complex of the ubiquitin (Ub) pathway for structural analysis. This pathway is a eukaryotic signalling system involving post-translational modification of target protein amino groups with Ub. The variety of modification provided by Ub and its interplay with other Small Ubiquitin-like Modifier (SUMO) signalling proteins controls an array of cellular responses. The pathway functions to activate the Ub C-terminus, forming highly reactive thioester conjugates between Ub and the active site cysteine of a series of enzymes: E1, E2 and in some cases E3. This thesis explores the use of an isopeptide linkage to stabilise an E2~Ub conjugate in complex with an E3 enzyme. Sample conditions were optimised for the future use of PELDOR spectroscopy to structurally analyse the E2~Ub alone and in complex with E3.