Interrogating and Predicting Tolerated Sequence Diversity in Protein Folds: Application to E. elaterium Trypsin Inhibitor-II Cystine-Knot Miniprotein
Figure 8
Trypsin-binding levels of EETI loop 3 predicted and randomly-generated clones.
Motif-filtered clones (dark grey), least-similar clones (light grey), randomly generated clones (white), negative control (potato carboxypeptidase inhibitor knottin, black), and EETIwt (striped) were individually displayed on the surface of yeast and analyzed by flow cytometry. Predicted clones are preceded with a ‘p’ while randomly-generated clones are preceded with an ‘r.’ Protein expression levels were quantified by immunofluorescence staining of the cMyc epitope tag. Retention of the knottin fold was determined by binding of fluorescently-labeled trypsin (25 nM). Trypsin binding levels were adjusted to account for differences in protein expression levels and then normalized to the trypsin-binding level of EETIwt. Trypsin-binding experiments were performed in triplicate and error bars denote standard deviations. Predicted clones showing statistically significant differences in trypsin binding levels compared to EETIwt are marked with an asterisk (*) or a double asterisk (**) to indicate lower and higher binding levels, respectively.