## 5 Results II: IFP family
### Monomerization of multimeric IFP  
Within the IFP, different monomer to tetramer transitions can occur. Here we focus on the one that is expected to characterize close homologs of the DsRed protein, Figure 1. A set of single position runs aiming at identifying putative SDPs that favor the monomeric character was performed as a first test of the procedure. The sequences were divided in two classes consisting of 26 monomeric and 66 multimeric proteins. Average identities were \(51.6\%\), \(73.6\%\) and \(49.9\%\) for the whole set, the first and the second class, respectively. Table 5 shows the 20 best ranking positions, estimated according to the different choices of similarity matrix described in the Methods. As in the previous section, for classification and assessment purposes, we associated a numeric code, \(S\), to each position. Consistent with the fact that experimentally validated positions can be categorized as belonging to the multimeric forming interface or not, \(S\) values are defined as: \(S\)=1 experimentally validated, at the multimeric-forming interface; \(S\)=2 experimentally validated, not at the multimeric-forming interface; \(S\)=3 untested, at the multimeric-forming interface; \(S\)=4 untested, not at the multimeric-forming interface. **Figure 2.** Alignment of a subset of 18 crystallized IFPs, labeled according to their pdb code. The sequences are separated according to their multimeric class and amino acids in specific positions colored as in Table 6 (complete alignment given in the SI). The whole IFP set has an overall average identity of \(40.8\%\). The sequence identity is larger in the first part of the sequence up to position \(\sim\)100, and smaller in the second half, except for isolated residues such as the highly conserved E197 (numbering according to DsRed). Monomeric  
2850 MVSKGRENNGAIIKKFM  
As shown in Table 5, different choices of the substitution matrix lead to similar performances, pointing to a good stability of the scheme. We believe that the (slightly) better performance of the Identity matrix is due to the presence in the set of several point mutants at some promising positions. This enrichment of local amino acid distribution brings a level of detail that can be better seized by a sharper discrimination between the different amino acids. Position 147 is assigned a star since it has been identified in experiments, but it can lead to non-fluorescent proteins [27, 30] and so it deserves a special classification. In Table 5 we also compared SDPhound results with those obtained by the SDPpred Web Server [2] using the same alignment and under the same conditions of shuffle number and of percentage of gaps exclusion. SDPpred returns a slightly lower number of hits and predicts only 9 of its positions to be statistically significant. Differences could arise from the different probability smoothing procedure and from the removal of background correlation, which does not seem to improve the results when performed on our set (see Sect. 4. of SI). The last row in the table reports how many of the 33 positions that are known to affect the aggregation state ranked higher than 33 in the various runs. As it can be seen from the Table, all runs succeed in pointing out the majority of the relevant positions with only minor fluctuations in the quality of the results. Looking for primary physical features  
Having established a preliminary ranking of the putative SDPs, we address the question of identifying the relevant physical property/ies at each position, by means of the pigeonholing procedure devised above. Based on the results of the previous runs, and in order to give a more clear-cut meaning to the pigeonholes, we again chose the Identity matrix in the definition of the substitution probability, see eq. (14). In Table 6 we traced the 20 best ranking positions found in the Id column of Table 5 to investigate whether they were high ranked in one or more of those \"per physical observable\" based runs. Let us review the most significant results.