The following paper is very important in connection with protein
evolvability:
Douglas D. Axe, "Extreme functional sensitivity to conservative amino
acid changes on enzyme exteriors", J.Mol.Biol. 301 (2000), 585-95.
It shows experimentally that by far the largest proportion of
conceivable evolutionary paths may not be neutral but deleterious.
Axe's abstract:
"Mutagenesis studies and alignments of homologous sequences have
demonstrated that protein function typically is compatible with a
variety of amino-acid residues at most exterior non-active-site
positions. These observations have led to the current view that
functional constraints on sequence are minimal at these positions. Here,
it is shown that this inference assumes that the set of acceptable
residues at each position is independent of the overall sequence
context. Two approaches are used to test this assumption. First, highly
conservative replacements of exterior residues, none of which would
cause significant functional disruption alone, are combined until
roughly one in five have been changed. This is found to cause complete
loss of function in vivo for two unrelated monomeric enzymes: barnase (a
bacterial RNase) and TEM-1 [beta]-lactamase. Second, a set of hybrid
sequences is constructed from the 50%-identical TEM-1 and Proteus
mirabilis [beta]-lactamases. These hybrids match the TEM-1 sequence
except for a region at the C-terminal end, where they are random
composites of the two parents. All of these hybrids are biologically
inactive. In both experiments, complete loss of activity demonstrates
the importance of sequence context in determining whether substitutions
are functionally acceptable. Contrary to the prevalent view, then,
enzyme function places severe constraints on residue identities at
positions showing evolutionary variability, and at exterior
non-active-site positions, in particular. Homologues sharing less than
about two-thirds sequence identity should probably be viewed as distinct
designs with their own sets of optimizing features."
This shows that previous estimates of the number of different possible
sequences that might show a given biological activity, such as H.P.
Yockey's ("Information theory and molecular biology" (Cambridge:
Cambridge Univ.Press, 1992), p.254), are likely to be vast overestimates
because they neglect possible interdependencies between different
amino-acid positions. I expressed this suspicion in my (unpublished)
paper at the 1988 Tacoma, WA, conference about Sources of Information
Content in DNA.
Peter Ruest <pruest@dplanet.ch>
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