Synthetic single domain antibodies for the conformational trapping of membrane proteins

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.


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Binder selections against target proteins were carried out with two technical replicates, giving very similar degrees of binder enrichment as judged by pPCR.
ELISA hits were replicated in a cross-specificity ELISA where the sybodies were tested against an array of target and dummy proteins.
qPCR runs were performed as technical triplicates and were highly reproducible. The obtained CT values were averages of these triplicates.
Thermal unfolding using Sypro Orange was performed by two technical replicates, which were highly reproducible. For fitting, representative datasets were used (see legend Figure 1- Figure Supplement 3).
SPR measurements on the MBP sybodies contained three technical replicates for each sybody concentration (see legend Figure 2- Figure Supplement 4). The TM287/288 sybodies were measured by SPR (Biorad ProteOn) with one replicate for each sybody concentration. SPR measurements for ENT1 and GlyT1 sybodies were carried out twice on different SPR chips, but using the same biological material and were highly reproducible. Representative data are shown in this case.
Each SPA-TS measurement (i.e. in presence or absence of syobdy) is based on 12 data points, which were in case of ENT1 determined as technical triplicates (error bars in Figure 5C are standard deviations) or in case of GlyT1 as single measurements.
ATPase activity measurements were carried out with three technical replicates for each data point (i.e. each sybody concentration) and these data were used to calculate the average and standard deviations (see legend Figure 3). Statistical reporting  Statistical analysis methods should be described and justified  Raw data should be presented in figures whenever informative to do so (typically when N per group is less than 10)  For each experiment, you should identify the statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures (e.g., mean, median, SD, SEM, confidence intervals; and, for the major substantive results, a measure of effect size (e.g., Pearson's r, Cohen's d)  Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.
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The experiments described in this paper did not require group allocation.
The structures of the sybody-MBP complexes were deposited on the protein database (PDB).