Probing unevolved sequence space for metal binding

Abstract

Metalloproteins constitute a third of all proteins and catalyze functions indispensable to life. In order to examine how early proteins acquired their metal-binding ability, a library of de novo, unevolved 4-helix bundle (i.e. proteins which have not been biased by natural selection) proteins was probed for their ability to bind cobalt, copper, and zinc ions. All proteins examined bound at least one cation with weak affinity, and 88.9% of those whose binding to more than one cation was investigated bound to one of the cations with at least moderate affinity. While the prevalence of metal-binding residues had no correlation with binding affinity, proteins predominantly bound the three cations in the order specified by the Irving-Williams series (Co < Zn < Cu). These results not only showcase the ability of this library to serve as a model for the evolution of protein function, but also suggest that cobalt-binding enzymes arose early in evolutionary history due to geochemical fluctuations in transition metal abundance, rather than due to an innate protein-binding ability.