Introducing SEC–SANS for studies of complex self-organized biological systems

Abstract

Small‐angle neutron scattering (SANS) is maturing as a method for studying complex biological structures. Owing to the intrinsic ability of the technique to discern between 1H‐ and 2H‐labelled particles, it is especially useful for contrast‐variation studies of biological systems containing multiple components. SANS is complementary to small‐angle X‐ray scattering (SAXS), in which similar contrast variation is not easily performed but in which data with superior counting statistics are more easily obtained. Obtaining small‐angle scattering (SAS) data on monodisperse complex biological structures is often challenging owing to sample degradation and/or aggregation. This problem is enhanced in the D2O‐based buffers that are typically used in SANS. In SAXS, such problems are solved using an online size‐exclusion chromatography (SEC) setup. In the present work, the feasibility of SEC–SANS was investigated using a series of complex and difficult samples of membrane proteins embedded in nanodisc particles that consist of both phospholipid and protein components. It is demonstrated that SEC–SANS provides data of sufficient signal‐to‐noise ratio for these systems, while at the same time circumventing aggregation. By combining SEC–SANS and SEC–SAXS data, an optimized basis for refining structural models of the investigated structures is obtained.

Circularized and solubility‐enhancedMSPs facilitate simple and high‐yield production of stable nanodiscs for studies of membrane proteins in solution

“…6A). This chromatogram is very similar to chromatograms obtained after incorporation of CorA in MSP1E3D1 nanodiscs [32], suggesting that nanodisc reconstitution of CorA is not particularly affected by the more restricted csMSP1E3D1.…”

Structure and dynamics of a nanodisc by integrating NMR, SAXS and SANS experiments with molecular dynamics simulations

“…SEC-SANS was performed at the D22 small-angle scattering diffractometer at the ILL, Grenoble, France using a very recently developed SEC-SANS setup. 35,36 Briefly, the setup was as follows: the in situ SEC was done using a modular HPLC system (Serlabo) equipped with a Superdex200 10/300 GL gel filtration column (GE) with a void volume of approxi- performed using the GRASP software. 37 The SANS data appropriate for buffer subtraction was identified based on when the 280 nm absorption during the SEC curve showed no trace of protein.…”

PSX, Protein–Solvent Exchange: software for calculation of deuterium-exchange effects in small-angle neutron scattering measurements from protein coordinates

“…These considerations become increasingly relevant for proteins with domains shielded from the solvent such as, for example, a membrane protein in a phospholipid bilayer nanodisc (Wadsä ter et al, 2012;Kynde et al, 2014;Johansen et al, 2018), liposomes (Rubinson et al, 2013), cubic lipid phase (van 't Hag et al, 2016), detergent micelles (Midtgaard et al, 2018), or proteins complexed with e.g. other proteins (Whitten et al, 2007;Clifton et al, 2012) or RNA (Gabel, 2015).…”

“…On a similar note, in-line size exclusion chromatography for BioSANS and stopped-flow time-resolved SANS (Rö ssle et al, 2000) have recently emerged as a promising technique for investigating particularly complicated and fragile biological samples of interest (Jordan et al, 2016;Johansen et al, 2018). When employing this technique, samples are exposed to the solvent, in many cases D 2 O, only minutes or even seconds before being irradiated by the neutron beam, meaning that the exchange process is far from equilibrium.…”