Premium
Circularized and solubility‐enhanced MSP s facilitate simple and high‐yield production of stable nanodiscs for studies of membrane proteins in solution
Author(s) -
Johansen Nicolai Tidemand,
Tidemand Frederik Grønbæk,
Nguyen Tam T. T. N.,
Rand Kasper Dyrberg,
Pedersen Martin Cramer,
Arleth Lise
Publication year - 2019
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14766
Subject(s) - nanodisc , circular dichroism , scaffold protein , chemistry , dispersity , membrane protein , biophysics , dynamic light scattering , membrane , crystallography , nanotechnology , materials science , biochemistry , biology , signal transduction , organic chemistry , nanoparticle
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 MSP 1E3D1, we designed an optimized membrane scaffold protein (His‐ls MSP 1E3D1) 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 (cs MSP 1E3D1) 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 (ls MSP 1E3D1) nanodiscs are structurally similar and have the expected nanodisc structure. Second, we show that ls MSP 1E3D1 nanodiscs are more stable compared to the template MSP 1E3D1 nanodiscs as an effect of the extra negative charges and that cs MSP 1E3D1 nanodiscs have further improved stability as an effect of circularization. Finally, we show that a membrane protein can be efficiently incorporated in cs MSP 1E3D1 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.