Structural comparison of in vitro evolved proteins with biological proteins will help determine the extent to which biological proteins sample the structural diversity available in protein sequence space. We have previously isolated a family of nonbiological ATP binding proteins from an unconstrained random sequence library. One of these proteins was further optimized for high-affinity binding to ATP, but biophysical characterization proved impossible due to poor solubility. To determine if such nonbiological proteins can be optimized for improved folding stability, we performed multiple rounds of mRNA-display selection under increasingly denaturing conditions. Starting from a pool of protein variants, we evolved a population of proteins capable of binding ATP in 3 M guanidine hydrochloride. One protein was chosen for further characterization. Circular dichroism, tryptophan fluorescence, and (1)H-(15)N correlation NMR studies show that this protein has a unique folded structure.