Date Published: December 01, 2018
Publisher: International Union of Crystallography
Author(s): Wanatchaporn Arunmanee, Richard K. Heenan, Jeremy H. Lakey.
Using the unique ability of small-angle neutron scattering to resolve a hydrogen-rich surfactant from a deuterated membrane protein, the results of removing free surfactant from the equilibrium are revealed.
Membrane proteins (MPs) play a vital role in cell function, and many of them, such as GPCRs and ion channels, have been exploited as drug targets. Therefore, over the years they have been the target of many structural and functional studies. Conventionally, when extracting MPs from biological membranes they must be handled in detergents in order to keep them soluble in aqueous solution. As detergents sometimes destabilize MPs, it is a formidable task to look for suitable detergents which maintain both their structure and function. To overcome this problem, several novel approaches have been developed to stabilize MPs in close-to-native environments (Hein et al., 2014 ▸). J.-L. Popot and coworkers invented a new class of detergents which are based upon an amphipathic polymer called ‘amphipol’ (APol; Tribet et al., 1996 ▸). APol comprises an anionic polyacrylate backbone partially and randomly derivatized with hydrophobic groups: octylamine and isopropylamine. APol makes multiple contacts with MPs, hence the affinity of MP for APol is high. In contrast to conventional detergents, APol is able to solubilize MPs in the near-absence of free APol (Tribet et al., 1997 ▸; Popot et al., 2003 ▸). Structural studies of MP in complex with APol have been carried out using several biophysical techniques such as electron microscopy (EM; see, for example, Cao et al., 2013 ▸; Liao et al., 2013 ▸; Lu et al., 2014 ▸; Fitzpatrick et al., 2017 ▸), small-angle neutron scattering (SANS; Gohon et al., 2008 ▸) and nuclear magnetic resonance (NMR; Zoonens et al., 2005 ▸; Catoire et al., 2010 ▸).
APols, a new class of detergents, have been used in a number of structural studies including NMR, SANS, EM etc. OmpF was reconstituted into APol with the aim of solubilizing and stabilizing OmpF in solution for molecular-interaction studies. Unexpectedly, instead of forming individual particles in solution, TEM data indicated that OmpF–APol assembled as filaments automatically after the removal of free APol by SEC (Arunmanee et al., 2014 ▸). This self-association of MP–APol complexes when lacking free APol has also been reported by Zoonens et al. (2007 ▸) and Gohon et al. (2008 ▸). This suggested that free APol is essential for the stability of MP–APol complexes in solution. Here, SANS experiments on OmpF–APol complexes purified by SEC confirmed that some of the APol that was initially bound to monodisperse OmpF immediately after SEC dissociated from the complex to create a new pool of free APol. Once this fraction of the APol had been removed from the OmpF–Apol complexes, the remaining APol was not sufficient to keep OmpF monodisperse. Subsequently, the filaments start to assemble rapidly, presumably to minimize the hydrophobic surface exposed to the aqueous buffer. The model generated from the SANS data also suggests that APol wraps around OmpF in a similar way to conventional detergents, so that the removal of Apol increases the exposure of the hydrophobic belt. The SANS experiment on these complexes was unable detect the filamentous structure observed by EM; the complexes appeared as distinct core shell structures. An upturn in the low-q region is an indication of a filamentous structure, but this was only observed in the sample in 100% D2O. The lack of this feature could be owing to the fact that the scattering of free APols is stronger than that in the filaments or that it is difficult to see them in the q-range of the SANS2D instrument. The OmpF filaments are easily disrupted by adding lipopolysaccharide (LPS) to OmpF–APol complexes. LPS, a lipid found in the outer leaflet of Gram-negative bacteria, specifically binds to the hydrophobic belt of OmpF (Arunmanee et al., 2016 ▸), suggesting again that the filaments are arranged as side-to-side strips of OmpF trimers. Interestingly, the addition of LPS leads to a sheet-like two-dimensional structure (Arunmanee et al., 2014 ▸) which is reminiscent of the outer membrane of E. coli comprising OmpF and LPS. Thus, MP–Apol filaments may even provide a method of creating two-dimensional crystals for structural studies (Baboolal et al., 2008 ▸; Arunmanee et al., 2014 ▸), with the minimal remaining Apol acting as a crystallization chaperone.