Small-angle X-ray and neutron scattering are well-established, non-invasive experimental techniques to interrogate global structural properties of biological membrane mimicking systems under physiologically relevant conditions. Recent developments, both in bottom-up sample preparation techniques for increasingly complex model systems, and in data analysis techniques have opened the path toward addressing long standing issues of biological membrane remodelling processes. These efforts also include emerging quantitative scattering studies on live cells, thus enabling a bridging of molecular to cellular length scales. Here, we review recent progress in devising compositional models for joint small-angle X-ray and neutron scattering studies on diverse membrane mimics -- with a specific focus on membrane structural coupling to amphiphatic peptides and integral proteins -- and live Escherichia coli. In particular, we outline the present state-of-the-art in small-angle scattering methods applied to complex membrane systems, highlighting how increasing system complexity must be followed by an advance in compositional modelling and data-analysis tools.

The flagellated Gram-negative bacteriumEscherichia coliis one of the most studied microorganisms. Despite extensive studies as a model prokaryotic cell, the ultrastructure of the cell envelope at the nanometre scale has not been fully elucidated. Here, a detailed structural analysis of the bacterium using a combination of small-angle X-ray and neutron scattering (SAXS and SANS, respectively) and ultra-SAXS (USAXS) methods is presented. A multiscale structural model has been derived by incorporating well established concepts in soft-matter science such as a core-shell colloid for the cell body, a multilayer membrane for the cell wall and self-avoiding polymer chains for the flagella. The structure of the cell envelope was resolved by constraining the model by five different contrasts from SAXS, and SANS at three contrast match points and full contrast. This allowed the determination of the membrane electron-density profile and the inter-membrane distances on a quantitative scale. The combination of USAXS and SAXS covers size scales from micrometres down to nanometres, enabling the structural elucidation of cells from the overall geometry down to organelles, thereby providing a powerful method for a non-invasive investigation of the ultrastructure. This approach may be applied for probingin vivothe effect of detergents, antibiotics and antimicrobial peptides on the bacterial cell wall.

We studied the transleaflet coupling of compositionally asymmetric liposomes in the fluid phase. The vesicles were produced by cyclodextrin-mediated lipid exchange and contained dipalmitoyl phosphatidylcholine (DPPC) in the inner leaflet and different mixed-chain phosphatidylcholines (PCs) as well as milk sphingomyelin (MSM) in the outer leaflet. In order to jointly analyze the obtained small-angle neutron and X-ray scattering data, we adapted existing models of trans-bilayer structures to measure the overlap of the hydrocarbon chain termini by exploiting the contrast of the terminal methyl ends in X-ray scattering. In all studied systems, the bilayer-asymmetry has large effects on the lipid packing density. Fully saturated mixed-chain PCs interdigitate into the DPPC-containing leaflet and evoke disorder in one or both leaflets. The long saturated acyl chains of MSM penetrate even deeper into the opposing leaflet, which in turn has an ordering effect on the whole bilayer. These results are qualitatively understood in terms of a balance of entropic repulsion of fluctuating hydrocarbon chain termini and van der Waals forces, which is modulated by the interdigitation depth. Monounsaturated PCs in the outer leaflet also induce disorder in DPPC despite vestigial or even absent interdigitation. Instead, the transleaflet coupling appears to emerge here from a matching of the inner leaflet lipids to the larger lateral lipid area of the outer leaflet lipids. Graphical abstract