This thesis covers multiple aspects of (synthetic) biochemistry. Starting from a global point of view and then going deeper into signalling on the single protein level. We investigate the energy balance of the cell and discuss several systems to (re)generate metabolic energy in a (synthetic) cell. We use one of these systems in the context of a cell-like environment and have developed a system in liposomes that is able to use external arginine as a fuel to regenerate ATP on the inside of the vesicle. We show basic physicochemical homeostasis and use the ATP that is produced to fuel one of the key proteins in osmoregulation, the ABC transporter OpuA. In case of an osmotic upshift, OpuA is activated by ionic strength and is able to import the compatible solute glycine betaine against large concentration gradients, powered by ATP. With use of single particle cryo-electron microscopy we have obtained a number of structures of OpuA in multiple conformations that help in understanding the transport mechanism and we show that OpuA is regulated by the second messenger cyclic-di-AMP, which acts as an emergency brake. Finally we demonstrate how we turned OpuA from a homodimeric into a heterodimeric protein complex and introduce an in silico approach to find new positions to label proteins that can be used for smFRET or DEER spectroscopy.
|Qualification||Doctor of Philosophy|
|Place of Publication||[Groningen]|
|Publication status||Published - 2020|