Membrane Interaction of Disease-Related Dynorphin A Variants

Abstract

The membrane interaction properties of two single-residue variants, R6W and L5S, of the 17-amino acid neuropeptide dynorphin A (DynA) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Corresponding gene mutations have recently been discovered in humans and causatively linked to a neurodegenerative disorder. The peptides were investigated in buffer and in isotropic solutions of q = 0.3 bicelles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or DMPC (0.8) and 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DMPG) (0.2). The CD results and the NMR secondary chemical shifts show that R6W-DynA has a small α-helical fraction in buffer, which increases in the presence of bicelles, while L5S-DynA is mainly unstructured under all conditions studied here. R6W-DynA has an almost complete association with zwitterionic bicelles (∼90%, as probed by NMR diffusion experiments), similar to the behavior of wtDynA, while L5S-DynA has a weaker association (∼50%). For all peptides, the level of bicelle association is increased in negatively charged bicelles. The L5A-DynA peptide adopts a very shallow position in the headgroup region of the bicelle bilayer, as studied by paramagnetic spin relaxation enhancement experiments using paramagnetic probes. Similarly, the results show that R6W-DynA is more deeply buried in the bilayer, with only the C-terminal residues exposed to solvent, again more similar to the case of wild-type DynA. We suggest that the results presented here may explain the differences in cell toxicity of these disease-related neuropeptide variants.

Characterization of fast‐tumbling isotropic bicelles by PFG diffusion NMR

“…Luchette and coworkers suspected that some mixing occurs also for q = 0.5 bicelles . Thorough investigations of the degree of segregation are only available for q = 0.5 bicelles, but it has been argued that bicelles persist down to q = 0.25 and lipid dynamics and size of such bicelles have been characterized . For q ‐values below 0.25, the separation of detergent rim and lipid bilayer is lost, and the aggregates formed are more appropriately described as mixed micelles .…”

“…Björnerås et al investigated the membrane association of two naturally occurring derivatives of dynorphin A, L5S‐DynA and R6W‐DynA, linked to a neurodegenerative disease. While L5S‐DynA does not perturb membranes, R6W‐DynA induces leakage in liposomes .…”

“…[25,29] [25] Also at 37 and 45°C DMPC/DHPC 0.5 300 5.7 25 5.2 [27] DMPC/DHPC 0.5 150 5.8 25 4.3 [85] With peptide DMPC/DHPC 0.54 250 7.4 15 5.6 [37] B. Chain length modification 12 : 0/DHPC 0.5 300 5.0 25 4.4 [25] Also at 37 and 45°C 16 : 0/DHPC 0.5 300 5.0 25 6.1 [25] Also at 37 and 45°C POPC/DHPC 0.53 * 6.5 27 6.34 [33] *Extrapolated to 0 concentration 18 : 1 PC/CHAPS 0.5 24 7.8 22 7.2 [86] 20 : 1 PC/CHAPS 0.5 24 7.8 22 6.4 [86] C. Head group modification [DMPC/MGDG 7 : 3]/DHPC 0.5 300 5.7 25 3.7 [27] Also for 10% MGDG [DMPC/DGDG 7 : 3]/DHPC 0.5 300 5.7 25 4.3 [27] Also [36] Preparation method 2 DMPC/D7PC 0.3 1.9 7.0 30 10.0 [36] Preparation method 2 DMPC/D8PC 0.3 1.8 7.0 30 4.7 [36] Preparation method 2 DMPC/cyclofos-6 0.3 7.1 7.0 30 6.8 [36] Preparation method 2 E. q-value modification DMPC/DHPC 0.3 150 5.7 25 6.3-6.7 [79] With peptide; also for [DMPC/DMPG 5 : 1]/DHPC POPC/DHPC 0.15 * 6.5 27 9.00 [33] *Extrapolated to 0 concentration F. Concentration modification DMPC/DHPC 0.3 238 7.0 30 9.5 [36] Preparation method 2 DMPC/DHPC 0.3 119 7.0 30 13.1 [36] Preparation method 2 DMPC/DHPC 0.3 59.4 7.0 30 14.4 [36] Preparation method 2 G. Temperature modification DMPC/DHPC 0.5 300 5.0 25 5.2 [25] r H = 4.7 nm DMPC/DHPC 0.5 300 5.0 37 6.4 [25] r H = 5.1 nm DMPC/DHPC 0.5 300 5.0 45 6.8 [25] This behavior is pronounced for bicelles with q ≥ 1. The sigmoidal size increase near the DMPC phase transition suggests that the bicelle size increases with increasing acyl chain mobility.…”

Elevated mutant dynorphin A causes Purkinje cell loss and motor dysfunction in spinocerebellar ataxia type 23

“…These mutant dynorphin A peptides displayed neurotoxic properties in cultured striatal neurons (Bakalkin et al, 2010), and induce pathological pain when injected into mice in femtomolar doses (Watanabe et al, 2012). Although the mechanism is unclear, recent studies indicate that these mutant peptides interact with and penetrate cellular membranes in vitro, and thus lead to cell death (Madani et al, 2011;Bjö rnerå s et al, 2013). Increased expression of PDYN and its neuropeptides have also been implicated in age-related decline of cognition and memory (Kotz et al, 2004;Ménard et al, 2013).…”

Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

“…pastoris membrane patches, the affinity of dynorphin A ( K i ) was enriched 20-fold when receptors were reconstituted in Nanodiscs. The increased affinity indicates that the lipid composition can be of importance for the interaction between KOR and the peptide agonist dynorphin A (1–17) [ 57 , 58 ].…”