Dynorphin A – Interactions with receptors and the membrane bilayer

Abstract: The work presented in this thesis concerns the dynorphin neuropeptides, and dynorphin A (DynA) in particular. DynA belongs to the wider class of typical opioid peptides that, together with the opioid receptors, a four-membered family of GPCR membrane proteins, form the opioid system. This biological system is involved or implicated in several physiological processes such as analgesia, addiction and depression, and effects caused by DynA through this system, mainly through interaction with the kappa subtype of the opioid receptors (KOR), are called the opioid effects. In addition to this, non-opioid routes of action for DynA have been proposed, and earlier studies have shown that direct membrane interaction is likely to contribute to these non-opioid effects. The results discussed here fall into either of two categories; the interaction between DynA and a fragment of KOR, and the direct lipid interaction of DynA and two variant peptides.For the receptor interaction case, DynA most likely causes its physiological effects through binding its N-terminal into a transmembrane site of the receptor protein, while the extracellular regions of the protein, in particular the extracellular loop II (EL2), have been shown to be important for modulating the selectivity of KOR for DynA. Here we have focussed on the EL2, and show the feasibility of transferring this sequence into a soluble protein scaffold. Studies, predominantly by nuclear magnetic resonance (NMR) spectroscopy, of EL2 in this new environment show that the segment has the conformational freedom expected of a disordered loop sequence, while the scaffold keeps its native beta-barrel fold. NMR chemical shift and paramagnetic resonance enhancement experiments show that DynA binds with high specificity to EL2 with a dissociation constant of approximately 30 micro Molar, while binding to the free EL2 peptide is an order of magnitude weaker. The strength of these interactions are reasonable for a receptor recognition event. No binding to the naked scaffold protein is observed.In the second project, the molecules of interest were two DynA peptide variants recently found in humans and linked to a neurological disorder. Previously published reports from our group and collaborators pointed at very different membrane-perturbing properties for the two variants, and here we present the results of a follow-up study, where the variants R6W-DynA and L5S-DynA were studied by NMR and circular dichroism (CD) spectroscopy in solutions of fast-tumbling phospholipid bicelles, and compared with wild type DynA. Our results show that R6W-DynA interacts slightly stronger with lipids compared to wild type DynA, and much stronger compared to L5S-DynA, in terms of bicelle association, penetration and structure induction. These results are helpful for explaining the differences in toxicity, membrane perturbation and relationship to disease, between the studied neuropeptides.