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This is because pairs of amino acids that interact with each other tend to 'co-evolve'; that is, if one amino acid changes, then the second amino acid also changes in order to accommodate it.
We then mapped the abnormal amino acid changes to the protein sequence of each gene and determined which domain had been altered.
The deleterious effects of amino acid changes were then predicted for proteins derived from the first transcript of each gene with both the SIFT (Ng and Henikoff, 2003, 2006) and MAPP (Stone and Sidow 2005) software packages.
If no amino acid change was found, then the residue was considered as "highly conserved".
If a gene is highly constrained by purifying selection (any amino acid change is deleterious) then one expects πns << πs.
This phenomenon was due to the interaction between ions and the enzyme surface charge which could markedly affect the ionization of some amino acid residues, then change the enzyme conformation, and alter the enzyme activity.
The evolutionary rate of amino acid change across species is then the number of amino acid substitutions divided by the total time elapse in the tree.
We then predicted the effect of the amino acid changes in GTF2H5 using SIFT tool [ 30], however, no intolerant changes were found among these disruptive mutations.
We then calculate the change in fitness for every single amino acid change, to generate, where is the fitness of an amino acid sequence differing from the reference sequence by the single replacement of for.
And then something changed.
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