Analysing DHPC/DMPC bicelles by diffusion NMR and multivariate decomposition.

Mixtures of lipids and detergents are known to form bicelles at certain parameter ranges, but many uncertainties remain concerning the details of the phase behaviour of these mixtures and the morphology of the formed lipid assemblies. Here we used nuclear magnetic resonance (NMR) diffusion data in combination with the multivariate processing method speedy component resolution (SCORE) to analyse mixtures of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the relative concentration q=[DMPC]/[DHPC]=0.5 at total lipid concentrations ranging from 15 to 300 mM. With this approach we were able to resolve the heavily overlapping mixture spectra into component spectra and obtained reliable diffusion coefficients for lipid concentrations in the range 15 to 300 mM, although at high concentrations (250-300 mM), non-negativity constraints or overfactoring was required to successfully decompose the data. At 50-300 mM total lipid concentration, the radii estimated from the diffusion coefficient of DMPC indicate assemblies of the appropriate bicelle size, although small size variations exist, while at lower concentrations the morphology appears to change to larger assemblies. Taken together, the results suggest that for q=0.5 DMPC/DHPC mixtures there is a relatively broad concentration range above 50 mM where bicelles may reliably be assumed to adopt the 'classical' bicelle morphology. The study clearly demonstrates the usefulness of our approach for accurately determining physical properties of complex mixtures such as bicelles. Both reliable diffusion coefficients and chemical shifts can be derived from overlapping data. This should prove useful for analysing the behaviour of other, more complex, lipid mixtures.

Resolving complex mixtures: trilinear diffusion data.

Complex mixtures are at the heart of biology, and biomacromolecules almost always exhibit their function in a mixture, e.g., the mode of action for a spider venom is typically dependent on a cocktail of compounds, not just the protein. Information about diseases is encoded in body fluids such as urine and plasma in the form of metabolite concentrations determined by the actions of enzymes. To understand better what is happening in real living systems we urgently need better methods to characterize such mixtures. In this paper we describe a potent way to disentangle the NMR spectra of mixture components, by exploiting data that vary independently in three or more dimensions, allowing the use of powerful algorithms to decompose the data to extract the information sought. The particular focus of this paper is on NMR diffusion data, which are typically bilinear but can be extended by a third dimension to give the desired data structure.

Membrane interaction of disease-related dynorphin A variants.

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.

Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the κ opioid receptor using a soluble protein scaffold.

The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the κ opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble ß-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.