Motivation: Current projects for the massive characterization of proteomes are generating protein sequences and structures with unknown function. The difficulty of experimentally determining functionally important sites calls for the development of computational methods. The first techniques, based on the search for fully conserved positions in multiple sequence alignments (MSAs), were followed by methods for locating family-dependent conserved positions. These rely on the functional classification implicit in the alignment for locating these positions related with functional specificity. The next obvious step, still scarcely explored, is to detect these positions using a functional classification different from the one implicit in the sequence relationships between the proteins. Here, we present two new methods for locating functional positions which can incorporate an arbitrary external functional classification which may or may not coincide with the one implicit in the MSA. The Xdet method is able to use a functional classification with an associated hierarchy or similarity between functions to locate positions related to that classification. The MCdet method uses multivariate statistical analysis to locate positions responsible for each one of the functions within a multifunctional family.

Results: We applied the methods to different cases, illustrating scenarios where there is a disagreement between the functional and the phylogenetic relationships, and demonstrated their usefulness for the phylogeny-independent prediction of functional positions.

Availability: All computer programs and datasets used in this work are available from the authors for academic use.


Supplementary information: Supplementary data are available at

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