Functionally important positions can comprise the majority of a protein's architecture

Concomitant with the genomic era, many bioinformatics programs have been developed to identify functionally important positions from sequence alignments of protein families. To evaluate these analyses, many have used the LacI/GalR family and determined whether positions predicted to be “important” are validated by published experiments. However, we previously noted that predictions do not identify all of the experimentally important positions present in the linker regions of these homologs. In an attempt to reconcile these differences, we corrected and expanded the LacI/GalR sequence set commonly used in sequence/function analyses. Next, a variety of analyses were carried out (1) for the entire LacI/GalR sequence set and (2) for a subset of homologs with functionally‐important “YxPxxxAxxL” motifs in their linkers. This strategy was devised to determine whether predictions could be improved by knowledge‐based sequence sorting and—for some analyses—did increase the number of linker positions identified. However, two functionally important linker positions were not reliably identified by any analysis. Finally, we compared the new predictions to all known experimental data for E . coli LacI and three homologous linkers. From these, we estimate that >50% of positions are important to the functions of the LacI/GalR homologs. In corollary, neutral positions might occur less frequently and might be easier to detect in sequence analyses. Although analyses have successfully guided mutations that partially exchange protein functions, a better experimental understanding of the sequence/function relationships in protein families would be helpful for uncovering the remaining rules used by nature to evolve new protein functions. Proteins 2011; © 2011 Wiley‐Liss, Inc.