Nanodiscs are membrane mimetics that consist of a protein belt surrounding a lipid bilayer, and are broadly used for characterization of membrane proteins. Here, we investigate the structure, dynamics and biophysical properties of two small nanodiscs, MSP1D1ΔH5 and ΔH4H5. We combine our SAXS and SANS experiments with molecular dynamics simulations and previously obtained NMR and EPR data to derive and validate a conformational ensemble that represents the structure and dynamics of the nanodisc. We find that it displays conformational heterogeneity with various elliptical shapes, and with substantial differences in lipid ordering in the centre and rim of the discs. Together, our results reconcile previous apparently conflicting observations about the shape of nanodiscs, and paves the way for future integrative studies of larger complex systems such as membrane proteins embedded in nanodiscs.

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.

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.

Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations.

Phospholipid nanodiscs have quickly become a widely used platform for studies of membrane proteins. However, the molecular self-assembly process that ultimately should place a membrane protein inside a nanodisc is not well understood. This poses a challenge for a successful high-yield reconstitution of general membrane proteins into nanodiscs. In the present work, the self-assembly process of POPC-MSP1D1 nanodiscs was carefully investigated by systematically modulating the reconstitution parameters and probing the effect with a small-angle X-ray scattering analysis of the resulting nanodiscs. First, it was established that nanodiscs prepared using the standard protocol followed a narrow but significant size distribution and that the formed nanodiscs were stable at room temperature over a time range of about a week. Systematic variation of the POPC/MSP1D1 stoichiometry of the reconstitution mixture showed that a ratio of less than 75:1 resulted in lipid-poor nanodiscs, whereas ratios of 75:1 and larger resulted in nanodiscs with constant POPC/MSP1D1 ratios of 60:1. A central step in the self-assembly process consists in adding detergent-absorbing resin beads to the reconstitution mixture to remove the reconstitution detergent. Surprisingly, it was found that this step did not play a significant role for the shape and stoichiometry of the formed nanodiscs. Finally, the effect of the choice of detergent used in the reconstitution process was investigated. It was found that detergent type is a central determining factor for the shape and stoichiometry of the formed nanodiscs. A significantly increasing POPC/MSP1D1 stoichiometry of the formed nanodiscs was observed as the reconstitution detergent type is changed in the order: Tween80, DDM, Triton X-100, OG, CHAPS, Tween20, and Cholate, but with no simple correlation to the characteristics of the detergent. This emphasizes that the detergents optimal for solution storage and crystallization of membrane proteins, in particular DDM, should not be used alone for nanodisc reconstitution. However, our data also show that when applying mixtures of the reconstitution detergent cholate and the storage detergents DDM or OG, cholate dominates the reconstitution process and nanodiscs are obtained, which resemble those formed without storage detergents.