MS# 2005-01-00890A (Russ et al.) Supplementary Online Materials.
Supplementary Figure Legends
Supplementary Figure 1: Binding specificity assays determined by the oriented peptide library
assay for (A) 28 natural WW domains and (B) 10 artificial WW domains designed using the SCA
matrix1. Binding is measured by ELISA using an HRP-assay, and the data are reported as fold
above background binding in the absence of peptide substrate. Peptide libraries are as described
in the Fig. 2 legend. Error bars show the standard deviation of 3-4 independent measurements.
Supplementary Figure 2: Saturation mutagenesis of the peptide ligands for the two major
functional classes of WW domains identified. A Natural group I (Nedd4.3, panel a) and a group
III (FE65.1, panel b) WW domain were assayed for binding to an array of immobilized peptides
representing all possible amino acid substitutions to a canonical group I (a) or group III (b)
peptide, respectively. The wild-type peptide sequence is given at the left of each blot, and each
row represents the binding to peptides where that position is mutated to every amino acid as
marked. Binding of CC43 (panel c) and CC20 (panel d) artificial WW domains to either a group
I (c), or group III (d) peptide array. The data show that artificial group I and group III WW
domains show the same pattern of sensitivity to mutation of ligand positions as natural WW
domains of the same specificity class.
Supplementary Figure 3: Thermodynamic double mutant cycles2, 3 in the WW domain Nedd4.3
(N39), measuring the energetic coupling between the T28A mutant and mutants at three other
sites within the network of co-evolving residues (L3A, E8A, and H23A). The thermodynamic
mutant cycle formalism is shown in Fig. 5B. Briefly, each corner of the box contains the
equilibrium dissociation constant for a group I peptide (EYPPYPPPPYPSG) and either wild-type,
single mutant, or double mutant WW proteins, measured through isothermal titration
calorimetery. Values represent the mean and standard deviation of at least three measurements,
reported in units of M.  is the coupling parameter as described in the text.
1.
2.
3.
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