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

Abstract

Recently, an enzymatic reaction was utilized to covalently link the N and C termini of membrane scaffold proteins to produce circularized nanodiscs that were more homogeneous and stable than standard nanodiscs. We continue this development and aim for obtaining high yields of stable and monodisperse nanodiscs for structural studies of membrane proteins by solution small‐angle scattering techniques. Based on the template MSP1E3D1, we designed an optimized membrane scaffold protein (His‐lsMSP1E3D1) with a sortase recognition motif and high abundance of solubility‐enhancing negative charges. With these modifications, we show that high protein expression is maintained and that the circularization reaction is efficient, such that we obtain a high yield of circularized membrane scaffold protein (csMSP1E3D1) and downstream circularized nanodiscs. We characterize the circularized protein and corresponding nanodiscs biophysically by small‐angle X‐ray scattering, size‐exclusion chromatography, circular dichroism spectroscopy, and light scattering and compare to noncircularized samples. First, we show that circularized and noncircularized (lsMSP1E3D1) nanodiscs are structurally similar and have the expected nanodisc structure. Second, we show that lsMSP1E3D1 nanodiscs are more stable compared to the template MSP1E3D1 nanodiscs as an effect of the extra negative charges and that csMSP1E3D1 nanodiscs have further improved stability as an effect of circularization. Finally, we show that a membrane protein can be efficiently incorporated in csMSP1E3D1 nanodiscs. Large‐scale production methods for circularized nanodiscs with improved thermal and temporal stability will facilitate better access to the nanodisc technology and enable applications at physiologically relevant temperatures.

Lipid-bound ApoE3 self-assemble into elliptical disc-shaped particles

“…Therefore, the observation are in line with our expectation. These areas also agree well with previously reported values for other types of nanodiscs with POPC [23,27,49] and DMPC [50,51].…”

Mg2+-dependent conformational equilibria in CorA: an integrated view on transport regulation

“…For SANS, CorA was produced and purified essentially as described elsewhere 48 . For studies in DDM, the N-terminal His-tag was cleaved by tobacco etch virus (TEV) protease before gel filtration, whereas for nanodiscs, it was cleaved after incorporation (see below).…”

Ab-initio determination of the shape of membrane proteins in a nanodisc

“…In practice, the fit is carried out using the WillItFit software (Pedersen et al, 2013). Furthermore, the height of the MSP, L b is often fixed to 25.8Å, as determined by NMR (Bibow et al, 2017;Johansen et al, 2019). Once the nanodisc parameters have been determined, a set of beads is used to retrieve the shape of the membrane protein inserted in the bilayer.…”