Immobilization of Transmembrane Receptors for SPR Kinetic Measurements by using SpyCatcher-SpyTag Complex

Membrane proteins are ideal drug targets due to their ubiquitous responsibilities in cellular signaling, substrate and ion transport, and ease of access due to their location in the cell. Membrane proteins are also notoriously difficult to study due to their hydrophobicity and flexible sections. Surface plasmon resonance (SPR) is an optical method that has offered multiple methodologies to study membrane proteins, including immobilizing these proteins by using detergents, histidine tagged receptors, and nanodiscs. However, each of these methods can introduce unreliable data for their own respective reasons because the immobilization method is not sufficient for membrane proteins. The concepts of nanodiscs make them inherently applicable to membrane proteins as they have sufficient support to behave in their native conformation. However, nanodiscs can be difficult to stabilize, therefore rendering any binding affinities unreliable. A new method proposed is using protein complexes to stabilize the nanodisc, which is where I present the SpyCatcher-SpyTag system being used in tandem with nanodiscs for successful SPR studies of various transmembrane receptors. Three different receptors and their respective ligands underwent SPR kinetic assays to test the applicability of the SpyCatcher-SpyTag nanodisc immobilization. The binding affinities compared with available results from other studies demonstrated robust applications to multiple protein systems, that can also be utilized with other instrumentation, providing a well-rounded analysis of membrane protein interactions.