Identifying Conformation-Selective Heavy-Chain-Only Antibodies Against Membrane Proteins by a Thermal-Shift Scintillation Proximity Assay

  • Peter Stohler
  • Roger J. P. DawsonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2127)


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.

Key words

Heavy-chain-only VHH Sybody Nanobody Glycine transporter 1 SLC6A9 Human GlyT1 Crystallography VHH profiling Cryo-electron microscopy Nanobody-enabled reverse pharmacology 



We thank Alain Gast for technical advice and assistance.


  1. 1.
    Bosworth N, Towers P (1989) Scintillation proximity assay. Nature 341:167–168CrossRefGoogle Scholar
  2. 2.
    Harder D, Fotiadis D (2012) Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay. Nat Protoc 7:1569–1578CrossRefGoogle Scholar
  3. 3.
    Zimmermann I, Egloff P et al (2018) Synthetic single domain antibodies for the conformational trapping of membrane proteins. elife 7:e34317CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Roche Pharma Research and Early Development, Therapeutic ModalitiesRoche Innovation Center Basel, F. Hoffmann-La Roche LtdBaselSwitzerland

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