Evolving Proteins of Novel Composition

The behavior of proteins can be altered significantly by the incorporation of noncanonical amino acids. Changes in spectroscopic properties, thermal stability, and molecular recognition behavior have been reported. In many cases, however, introduction of novel amino acids causes loss of protein function. Herein we describe a new approach to protein engineering in which amino acid replacement is combined with directed evolution to create functional proteins of novel composition. Global replacement of the leucine residues of chloramphenicol acetyltransferase (CAT) by 5',5',5'-trifluoroleucine (TFL) results in a 20-fold reduction in the half-life (t_(1/2)) of thermal inactivation of the enzyme at 608C. Two rounds of random mutagenesis and screening yielded a variant of CAT containing three amino acid substitutions, which in fluorinated form demonstrates a 27-fold improvement in t_(1/2), recovering the loss in thermostability caused by fluorination.