Analysis and prediction of functional sub-types from protein sequence alignments

J Mol Biol. 2000 Oct 13;303(1):61-76. doi: 10.1006/jmbi.2000.4036.


The increasing number and diversity of protein sequence families requires new methods to define and predict details regarding function. Here, we present a method for analysis and prediction of functional sub-types from multiple protein sequence alignments. Given an alignment and set of proteins grouped into sub-types according to some definition of function, such as enzymatic specificity, the method identifies positions that are indicative of functional differences by comparison of sub-type specific sequence profiles, and analysis of positional entropy in the alignment. Alignment positions with significantly high positional relative entropy correlate with those known to be involved in defining sub-types for nucleotidyl cyclases, protein kinases, lactate/malate dehydrogenases and trypsin-like serine proteases. We highlight new positions for these proteins that suggest additional experiments to elucidate the basis of specificity. The method is also able to predict sub-type for unclassified sequences. We assess several variations on a prediction method, and compare them to simple sequence comparisons. For assessment, we remove close homologues to the sequence for which a prediction is to be made (by a sequence identity above a threshold). This simulates situations where a protein is known to belong to a protein family, but is not a close relative of another protein of known sub-type. Considering the four families above, and a sequence identity threshold of 30 %, our best method gives an accuracy of 96 % compared to 80 % obtained for sequence similarity and 74 % for BLAST. We describe the derivation of a set of sub-type groupings derived from an automated parsing of alignments from PFAM and the SWISSPROT database, and use this to perform a large-scale assessment. The best method gives an average accuracy of 94 % compared to 68 % for sequence similarity and 79 % for BLAST. We discuss implications for experimental design, genome annotation and the prediction of protein function and protein intra-residue distances.

MeSH terms

  • Adenylyl Cyclases / chemistry
  • Adenylyl Cyclases / metabolism
  • Algorithms
  • Amino Acid Sequence
  • Animals
  • Computational Biology / methods*
  • Databases as Topic
  • Entropy
  • Guanylate Cyclase / chemistry
  • Guanylate Cyclase / metabolism
  • Humans
  • L-Lactate Dehydrogenase / chemistry
  • L-Lactate Dehydrogenase / metabolism
  • Malate Dehydrogenase / chemistry
  • Malate Dehydrogenase / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Kinases / chemistry
  • Protein Kinases / metabolism
  • Proteins / chemistry*
  • Proteins / classification
  • Proteins / metabolism*
  • Sensitivity and Specificity
  • Sequence Alignment / methods*
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / metabolism
  • Software
  • Structure-Activity Relationship
  • Substrate Specificity


  • Proteins
  • L-Lactate Dehydrogenase
  • Malate Dehydrogenase
  • Protein Kinases
  • Serine Endopeptidases
  • Adenylyl Cyclases
  • Guanylate Cyclase