False occurrences of functional motifs in protein sequences highlight evolutionary constraints

Here we analyse the occurrence of functional motifs in random sequences and compare it to that observed in biological proteomes; the behaviour of random motifs is also studied. Most motifs exhibit a number of false positives significantly similar to the number of times they appear in randomized proteomes (=expected number of false positives). Interestingly, about 3% of the analysed motifs show a different kind of behaviour and appear in biological proteomes less than they do in random sequences. In some of these cases, a mechanism of evolutionary negative selection is apparent; this helps to prevent unwanted functionalities which could interfere with cellular mechanisms.Our thorough statistical and biological analysis showed that there are several mechanisms and evolutionary constraints both of which affect the appearance of functional motifs in protein sequences.The detection of functional sequence patterns (motifs) in a yet uncharacterized protein is one of the most widely used and powerful methods for assigning a function to proteins in newly sequenced proteomes. Sequence patterns associated to functional motifs are usually generated manually in an attempt to maximize the number of sequences that clearly belong to the set functionally characterized by the motif (true positives, TP), while minimizing the number of unrelated sequences (false positives, FP) [1]. The possibility of estimating the number of false predictions is critical in evaluating the significance of finding a pattern in a protein sequence. The false positive rate of a pattern on a large protein database can be estimated from the number of matches expected to occur by chance [2,3]. In the following we will refer to this thesis as the 'classical hypothesis'. Both these authors measured the expectation of occurrence of PROSITE [4] functional patterns in a sequence database of size N, simply as the product of N and the amino acid probabilities in each position of the pattern. The individual amino acid