Circularized and solubility‐enhanced MSPs facilitate simple and high‐yield production of stable nanodiscs for studies of membrane proteins in solution. (12th February 2019)

• Record Type: Journal Article
• Authors: Johansen, Nicolai Tidemand; Tidemand, Frederik Grønbæk; Nguyen, Tam T. T. N.; Rand, Kasper Dyrberg; Pedersen, Martin Cramer; Arleth, Lise
• Is Part Of: FEBS journal. Volume 286:Number 9(2019)
• Page Start: 1734
• Page End: 1751
• Publication Date: 2019-02-12
• Subjects: membrane protein -- nanodisc -- protein circularization -- small‐angle X‐ray scattering -- thermal and temporal stability; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.14766
• Languages: English
• ISSNs: 1742-464X

Abstract

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. Abstract : A solubility‐enhanced membrane scaffold protein was genetically designed by introducing high abundance of negatively charged amino acids. Its enhanced solubility facilitates straight‐forward and high‐yield expression, purification, and circularization. Nanodiscs made with this protein are highly conformationally stable and thus suitable for membrane protein incorporation and studies at physiologically relevant temperatures.