Sybodies for structural biology of membrane proteins

Membrane transporters are difficult to crystallize due to their limited solvent-exposed surface. In addition, the intrinsic flexibility of transporters impedes their analysis by cryo-EM. In vitro selected binding proteins offer the possibility to overcome both shortcomings at the same time: they can stabilize a transporter in a defined conformational state and mediate crystal contacts. We have previously used designed ankyrin repeat proteins (DARPins) as binder scaffold to study the structure and function of ABC exporters (Protein Science, Seeger et al. 2013; JBC, Mittal et al. 2012, Plos One, Seeger et al. 2012). Currently, we focus on single domain antibodies called nanobodies. Traditionally, nanobodies are obtained from immunized camelids such as alpacas (performed at the UZH Nanobody Service Facility).

In the past four years, we have established a platform for the selection of synthetic nanobodies called sybodies entirely in vitro (eLife, Zimmermann et al. 2018). To this end, we have constructed three sybody libraries exhibiting different randomized shapes (see picture). The sybody platform is ideally suited to select binders against delicate membrane proteins in the presence of non-covalent ligands. Thereby, sybodies trapping the targeted membrane protein in a defined conformational state are obtained. We generated sybodies which capture the ABC transporter TM287/288 in its outward-facing state for crystal structure determination (Hutter et al., in preparation). In the context of a CTI project in collaboration with Roche, we achieved to generate sybodies against two disease-relevant human SLC transporters, which were previously intractable to nanobody generation by immunization strategies (Sybody Platform, Contributed by Roger Dawson (PDF, 586 KB)). We continue to collaborate with Roche to jointly develop sybodies against disease-relevant targets. Recently, we started a CTI project in collaboration with leadXpro aiming at the generation of sybodies against bacterial membrane proteins. Further, we generate sybodies as pathogen capture reagents for rapid diagnostics of blood stream infections in a project funded by the NRP 72 "Antimicrobial Resistance".