Small‐angle X‐ray and neutron scattering demonstrates that cell‐free expression produces properly formed disc‐shaped nanolipoprotein particles. (13th February 2018)

• Record Type: Journal Article
• Authors: Cleveland, Thomas E.; He, Wei; Evans, Angela C.; Fischer, Nicholas O.; Lau, Edmond Y.; Coleman, Matthew A.; Butler, Paul
• Is Part Of: Protein science. Volume 27:Number 3(2018)
• Page Start: 780
• Page End: 789
• Publication Date: 2018-02-13
• Subjects: NLPs -- nanodiscs -- nanolipoprotein particles -- SANS -- SAXS -- small‐angle scattering -- cell‐free expression; Proteins -- Periodicals; 572.6
• DOI: 10.1002/pro.3365
• Languages: English
• ISSNs: 0961-8368

Abstract

Abstract: Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native‐like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent‐solubilized protein‐lipid mixture. Recently, an alternative method has been developed using direct cell‐free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell‐free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell‐free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core‐shell cylinder type model to all SANS/SAXS data simultaneously.