Towards more rational approaches of membrane protein stabilisation and novel structures of membrane-bound pyrophosphatase

Membrane proteins have a range of crucial biological functions and are targeted by most prescribed drugs despite lagging behind soluble proteins when it comes to their biochemical and biophysical characterisation. A major bottleneck in membrane protein research is protein instability upon extraction. Protein stabilisation strategies are typically expensive and labour-intensive. Therefore, I contributed to the development and evaluation of two new general-purpose tools, both designed for the streamlined and rational stabilisation of membrane proteins. The first tool, the integral membrane protein stability selector (IMPROvER), predicts stabilising point-mutations in membrane proteins using three individual approaches with additive prediction power. The second tool, a novel pre-prepared and easy-to-use screen for the high-throughput identification of stabilising lipids, facilitates the structural and functional analysis of stable and physiologically relevant protein sample. Both tools were successfully employed to stabilise a range of membrane proteins with different folds, topologies and modes of action at significantly reduced cost and work effort. Moreover, engineered or natively thermostable membrane-bound pyrophosphatases (M-PPase), were studied in more detail using conventional and time-resolved X-ray crystallography. Based on structural data obtained on a pyrophosphate-energised K+-independent H+-pump, I derived an updated model of ion-selectivity that is centred on a glutamate-serine interplay at the ion-gate. This is the first model that explains ion selectivity in all M PPase subclasses when considering functional asymmetry. Indeed, complementary time-resolved structural studies of a K+-dependent Na+-pump revealed asymmetric substrate binding to M-PPase active sites. These findings give valuable mechanistic insights into key processes of M-PPase biochemistry, which are of upmost importance for structure-guided drug discovery. Ultimately, tweaking M-PPase function has the potential to address existing and emerging challenges to human health and global food security as M-PPases play a vital role in the stress resistance of pathogens or salt and drought resistance in plants.