  BACKGROUND: Many of the mutations accumulated by naturally evolving proteins
are neutral in the sense that they do not significantly alter a protein's
ability to perform its primary biological function. However, new protein
functions evolve when selection begins to favor other, "promiscuous" functions
that are incidental to a protein's biological role. If mutations that are
neutral with respect to a protein's primary biological function cause
substantial changes in promiscuous functions, these mutations could enable
future functional evolution.
  RESULTS: Here we investigate this possibility experimentally by examining how
cytochrome P450 enzymes that have evolved neutrally with respect to activity on
a single substrate have changed in their abilities to catalyze reactions on
five other substrates. We find that the enzymes have sometimes changed as much
as four-fold in the promiscuous activities. The changes in promiscuous
activities tend to increase with the number of mutations, and can be largely
rationalized in terms of the chemical structures of the substrates. The
activities on chemically similar substrates tend to change in a coordinated
fashion, potentially providing a route for systematically predicting the change
in one function based on the measurement of several others.
  CONCLUSIONS: Our work suggests that initially neutral genetic drift can lead
to substantial changes in protein functions that are not currently under
selection, in effect poising the proteins to more readily undergo functional
evolution should selection "ask new questions" in the future.
