Abstract
Detergent micelles can solubilize membrane proteins, but there is always a need for a pool of free detergent at the critical micellar concentration to maintain the micelle-monomer equilibrium. Amphipol polymeric surfactants (APols) have been developed to replace conventional detergents in membrane-protein studies, but the role of free amphipol is unclear. It has previously been shown that the removal of free APol causes monodisperse outer membrane protein F (OmpF) to form long filaments. However, any remaining APol could not be resolved using electron microscopy. Here, small-angle neutron scattering with isotope contrast matching was used to separately determine the distributions of membrane protein and amphipol in a mixed sample. The data showed that after existing free amphipol had been removed from monodisperse complexes, a new equilibrium was established between protein-amphipol filaments and a pool of newly liberated free amphipol. The filaments consisted of OmpF proteins surrounded by a belt of Apol, whilst free oblate spheroid micelles of Apol were also present. No indications of long-range order were observed, suggesting a lack of defined structure in the filaments.
Highlights
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
Instead of forming individual particles in solution, transmission electron microscopy (TEM) data indicated that outer membrane protein F (OmpF)–Amphipol polymeric surfactants (APols) assembled as filaments automatically after the removal of free APol by size-exclusion chromatography (SEC) (Arunmanee et al, 2014)
smallangle neutron scattering (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
Summary
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. Over the years they have been the target of many structural and functional studies. 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). Popot and coworkers invented a new class of detergents which are based upon an amphipathic polymer called ‘amphipol’ (APol; Tribet et al, 1996). 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), smallangle neutron scattering (SANS; Gohon et al, 2008) and
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