Studying integral membrane protein by SANS using stealth reconstitution systems.

Abstract

Structural studies of integral membrane proteins (IMPs) are challenging as many of them require a lipid environment for full activity and stability. Reconstitution of IMPs into carrier systems such as nanodiscs or Salipro that mimic the native lipidic environment allow structural studies of membrane proteins in solution. The difficulty with this approach when applied to scattering techniques is the contribution of the carrier system to the scattering intensity and the subsequent challenging data analysis. Recently, so-called stealth carrier systems have been developed and applied to small-angle neutron scattering (SANS) studies of integral membrane proteins that become invisible to neutrons due to specific deuteration and solvent contrast-variation. In this chapter, we describe in detail how the well-studied ATP-binding cassette (ABC) transporter protein MsbA can be reconstituted into stealth nanodiscs and subsequently be studied by SANS. This approach allows for a direct observation of the scattering signal from MsbA without the contribution of the surrounding carrier system and enables detection of different conformational states. The protocols can also be adapted to other stealth carrier systems (such as stealth Salipro).