TY - JOUR TI - Synthetic single domain antibodies for the conformational trapping of membrane proteins AU - Zimmermann, Iwan AU - Egloff, Pascal AU - Hutter, Cedric AJ AU - Arnold, Fabian M AU - Stohler, Peter AU - Bocquet, Nicolas AU - Hug, Melanie N AU - Huber, Sylwia AU - Siegrist, Martin AU - Hetemann, Lisa AU - Gera, Jennifer AU - Gmür, Samira AU - Spies, Peter AU - Gygax, Daniel AU - Geertsma, Eric R AU - Dawson, Roger JP AU - Seeger, Markus A A2 - Forrest, Lucy VL - 7 PY - 2018 DA - 2018/05/24 SP - e34317 C1 - eLife 2018;7:e34317 DO - 10.7554/eLife.34317 UR - https://doi.org/10.7554/eLife.34317 AB - 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. KW - membrane protein KW - nanobody KW - ribosome display KW - phage display KW - in vitro selection KW - conformational trapping JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -