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Non-ionic detergent assists formation of supercharged nanodiscs and insertion of membrane proteins

DOI: 10.1016/j.bbamem.2022.183884 DOI Help

Authors: Frederik G. Tidemand (University of Copenhagen) , Sara Blemmer (University of Copenhagen) , Nicolai T. Johansen (University of Copenhagen) , Lise Arleth (University of Copenhagen) , Martin Cramer Pedersen (University of Copenhagen)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochimica Et Biophysica Acta (bba) - Biomembranes , VOL 1864

State: Published (Approved)
Published: June 2022
Diamond Proposal Number(s): 23041 , 27887

Open Access Open Access

Abstract: Nanodiscs are used to stabilize membrane proteins in a lipid environment and enable investigations of the function and structure of these. Membrane proteins are often only available in small amounts, and thus the stability and ease of use of the nanodiscs are essential. We have recently explored circularizing and supercharging membrane scaffolding proteins (MSPs) for nanodisc formation and found increased temporal stability at elevated temperatures. In the present study, we investigate six different supercharged MSPs and their ability to form nanodiscs: three covalently circularized and the three non-circularized, linear versions. Using standard reconstitution protocols using cholate as the reconstitution detergent, we found that two of the linear constructs formed multiple lipid-protein species, whereas adding n-Dodecyl-B-D-maltoside (DDM) with the cholate in the reconstitution gave rise to single-species nanodisc formation for these MSPs. For all MSPs, the formed nanodiscs were analyzed by small-angle X-ray scattering (SAXS), which showed similar structures for each MSP, respectively, suggesting that the structures of the formed nanodiscs are independent of the initial DDM content, as long as cholate is present. Lastly, we incorporated the membrane protein proteorhodopsin into the supercharged nanodiscs and observed a considerable increase in incorporation yield with the addition of DDM. For the three circularized MSPs, a single major species appeared in the size exclusion chromatography (SEC) chromatogram, suggesting monodisperse nanodiscs with proteorhodopsin incorporated, which is in strong contrast to the samples without DDM showing almost no incorporation and high polydispersity.

Journal Keywords: Nanodiscs; DDM; Proteorhodopsin; Self-assembly; Cholate; Small-angle scattering; Size exclusion chromatography

Subject Areas: Biology and Bio-materials

Instruments: B21-High Throughput SAXS

Added On: 22/02/2022 09:33


Discipline Tags:

Soft condensed matter physics Nanoscience/Nanotechnology Biophysics Life Sciences & Biotech

Technical Tags:

Scattering Small Angle X-ray Scattering (SAXS)