Biophysical studies of membrane associated biomolecular interactions

Abstract: Membrane associated interactions play important roles in various biological processes. The aim of this thesis is to study two types of membrane associated interactions by using biophysical methods, particularly NMR. First, the interaction of a targeting peptide and a receptor attached to membrane has been studied. Secondly, we were interested in the interaction between a galactosyltransferase and the involved membrane lipids, galactolipids, and thus novel membrane mimetic models containing galactolipids have been studied. In addition, membrane mimetic models, bicelles, under different conditions have been characterized. The targeting peptide studied here is a dual targeting peptide, ThrRS-dTP(2-60), which is capable of targeting a t-RNA synthase, ThrRS, to both the mitochondrial matrix and the chloroplast stroma. Tom20 is the first component in the mitochondrial outer membrane import machinary that interacts with ThrRS-dTP(2-60). Biophysical studies in this thesis on the dTP-Tom20 interaction showed that it shares several features with the interaction between mitochondrial targeting peptides and Tom20. An amphiphilic helix with a fccff motif from ThrRS-dTP(2-60) is crucial for the interaction with Tom20 and more than one region was involved. However, a relatively long C-terminal part of the peptide was not affected by the interaction with Tom20.   Novel membrane mimetic media were developed by introducing the galactolipids MGDG and DGDG into DMPC/DHPC bicelles. Diffusion NMR results indicated that there is no phase separation upon incorporation of as much as 30% galactoplids. The sizes of the new bicelles were, however, larger than DMPC/DHPC bicelles. 13C relaxation measurements showed that galactolipids in the bicelles have two distinct local dynamic regions: the galactose units together with the glycerol linker region, with limited local motion, and the acyl chains with extensive motion. The bicelles with galactolipids are suitable for studies of e.g. the interaction between the galactosyltransferases and galactolipids. Additionally, studies on the morphology of DMPC/DHPC mixtures under various conditions were performed. The size distribution for small, fast-tumbing bicelles was observed to be dependent on the q-value (the mole ratio between the long-chained lipids and the short-chained lipids), temperature and the total lipid concentration. With larger q-values and low concentrations, the DMPC/DHPC mixture was not monodisperse, as additional structures were observed in both dynamic light scattering and cryogenic transmission electron microscopy experiments. Hence, care is needed when using bicelles under new conditions.