Probing Membrane Protein Assembly into Nanodiscs by In Situ Dynamic Light Scattering: A(2A) Receptor as a Case Study

SIMPLE SUMMARY: Located within the biological cell membranes, integral membrane proteins are responsible for a large variety of vital cellular processes. In humans, nearly a quarter of the genome codes integral membrane proteins, therefore malfunction of these proteins is associated with a variety of symptoms and diseases such as obesity, cancer and Parkinson’s disease. Clearly, knowledge of membrane proteins behaviour, in both structural and functional terms, is important not only in medicine but also in the design of better drugs with improved pharmaceutical properties. Nevertheless, much still remains unknown about these proteins, mainly because of the technical challenges associated with their production and stability in vitro once removed from their native lipidic environment. Recently, several membrane mimetic systems have been developed including nanodisc lipid particles. Nanodiscs are self-assembled lipidic structures that “trap” membrane proteins into a disc shaped phospholipid bilayer that is stabilised by a belt made of a protein know as membrane scaffold protein (MSP). Membrane proteins assembled into lipidic nanodiscs can maintain their structural and functional integrity and are compatible with most biophysical methods. Here we demonstrate the use of in situ dynamic light scattering as a high-throughput screening tool to assess the best conditions for nanodisc assembly and protein incorporation. ABSTRACT: Membrane proteins play a crucial role in cell physiology by participating in a variety of essential processes such as transport, signal transduction and cell communication. Hence, understanding their structure–function relationship is vital for the improvement of therapeutic treatments. Over the last decade, based on the development of detergents, amphipoles and styrene maleic-acid lipid particles (SMALPs), remarkable accomplishments have been made in the field of membrane protein structural biology. Nevertheless, there are still many drawbacks associated with protein–detergent complexes, depending on the protein in study or experimental application. Recently, newly developed membrane mimetic systems have become very popular for allowing a structural and functional characterisation of membrane proteins in vitro. The nanodisc technology is one such valuable tool, which provides a more native-like membrane environment than detergent micelles or liposomes. In addition, it is also compatible with many biophysical and biochemical methods. Here we describe the use of in situ dynamic light scattering to accurately and rapidly probe membrane proteins’ reconstitution into nanodiscs. The adenosine type 2A receptor (A(2A)R) was used as a case study.