ABSTRACT
Over the last decades, the use of heavy-chain-only antibodies has received growing attention in academia and industry as research and diagnostic tools as well as therapeutics. Their generation has improved with the help of innovative new methods such as the sybody technology; however, identifying conformation-selective compounds against membrane proteins remains a major challenge. In this chapter, we apply a thermal shift scintillation proximity assay (SPA-TS) to identify sybodies from an in vitro display campaign with the ability to selectively stabilize the inhibitor-bound conformation of the human solute carrier (SLC) family transporter SC6A9 (GlyT1). Using detergent-purified GlyT1 protein and a tritium-labeled glycine uptake inhibitor small molecule, we find sybody candidates that increase the apparent melting temperature in SPA-TS by several degrees. The thermal shift stabilizes the GlyT1-inhibitor complex and qualifies the sybodies for structural studies and inhibitor-selective small molecule screening assays. The SPA-TS assay in its current form is adaptable to any antibody discovery campaign for membrane proteins and permits the generation of highly valuable tools in most stages of drug discovery and development.
Subject(s)
Biological Assay/methods , Immunoglobulin Heavy Chains/isolation & purification , Membrane Proteins/chemistry , Membrane Proteins/immunology , Animals , Antibody Specificity , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Glycine/metabolism , Glycine Plasma Membrane Transport Proteins/chemistry , Glycine Plasma Membrane Transport Proteins/immunology , Glycine Plasma Membrane Transport Proteins/metabolism , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/metabolism , Kinetics , Membrane Proteins/metabolism , Protein Binding , Protein Conformation , Substrate Specificity , Temperature , ThermodynamicsABSTRACT
Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.
