The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22-25 comprising a beta-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22-25 tetrapeptide sequences and presented these proteins on the outer membrane of Escherichia coli cells via fusion to the Iga(beta) autotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1-5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in beta-turns. The folding pathway of two selected variants, EETI-beta(NEDE) and EETI-beta(TNNK), was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.