FamClash: a method for ranking the activity of engineered enzymes

Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4142-7. doi: 10.1073/pnas.0400065101. Epub 2004 Feb 23.


This article introduces the computational procedure FamClash for analyzing incompatibilities in engineered protein hybrids by using protein family sequence data. All pairs of residue positions in the sequence alignment that conserve the property triplet of charge, volume, and hydrophobicity are first identified, and significant deviations are denoted as residue-residue clashes. This approach moves beyond earlier efforts aimed at solely classifying hybrids as functional or nonfunctional by correlating the rank ordering of these hybrids based on their activity levels. Experimental testing of this approach was performed in parallel to assess the predictive ability of FamClash. As a model system, single-crossover ITCHY (incremental truncation for the creation of hybrid enzymes) libraries were prepared from the Escherichia coli and Bacillus subtilis dihydrofolate reductases, and the activities of functional hybrids were determined. Comparisons of the predicted clash map as a function of crossover position revealed good agreement with activity data, reproducing the observed V shape and matching the location of a local peak in activity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacillus subtilis / enzymology
  • Computational Biology
  • Escherichia coli / enzymology
  • Peptide Library
  • Protein Engineering*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Tetrahydrofolate Dehydrogenase / genetics
  • Tetrahydrofolate Dehydrogenase / metabolism*


  • Peptide Library
  • Recombinant Fusion Proteins
  • Tetrahydrofolate Dehydrogenase