gI2.5.3 and rI4.5.1 framework mutations. (a) P51, T58 and I88 are presented in the wild-type Fn3 structure 1TTG (Main et al., 1992). (b) S60 is presented in the wild-type Fn3 structure. E98 was not included in this structure, but the C-terminal amino acid of the structure, T94, is indicated. (c) The indicated clone, with a c-myc epitope tag, was displayed on the surface of yeast with 37° induction. Cells were labeled with chicken anti-c-myc antibody followed by bovine anti-chicken phycoerythrin conjugate and analyzed by flow cytometry. The phycoerythrin signals above background were normalized to the gI2.5.3 sample and are presented as Fn3 per cell. Cells were labeled with 10 nM goat IgG-FITC conjugate and analyzed by flow cytometry. The FITC signal above background was divided by the relative number of Fn3 per cell, normalized to the gI2.5.3 sample, and is presented as IgG binding. Values are mean ± standard deviation of at least duplicate experiments. (d) As in (c) except samples were labeled with 100 pM rabbit IgG and were normalized to rI4.5.1.