Influence of Global Fluorination on Chloramphenicol Acetyltransferase Activity and Stability Tatyana Panchenko, 1 Wan Wen Zhu, 1 Jin Kim Montclare 1,2 1 Department of Chemical and Biological Sciences, Polytechnic University, 6 Metrotech Center, Brooklyn, New York 11201; telephone: 718-260-3679; fax: 718-260-3125; e-mail: jmontcla@poly.edu 2 Department of Biochemistry, SUNY-Downstate Medical Center, Brooklyn, New York 11203 Received 28 December 2005; accepted 2 March 2006 Published online 17 March 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20910 Abstract: Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5 0 ,5 0 ,5 0 -trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient tem- peratures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved under standard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemost- ability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced second- ary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability. ß 2006 Wiley Periodicals, Inc. Keywords: protein engineering; trifluoroleucine; activ- ity; thermostability; non-natural amino acid; residue- specific incorporation INTRODUCTION Protein engineering via rational design or laboratory evolution has been employed to enhance protein activity and stability, alter substrate specificities, elucidate function, and generate novel protein-based materials (Arnold, 1993, 1996; Dolgikh et al., 1996; Fersht and Winter, 1992; Petka et al., 1998; Yu and Tirrell, 2000). In vivo engineering has been dominated by mutagenesis where proteins are allowed to sample one of the natural sets of 20 amino acids. The ability to incorporate amino acid analogs into proteins beyond this available collection of 20 expands our capacity to manipulate the structure and function of proteins (Budisa, 2004; Link et al., 2003; Wang and Schultz, 2004). Various approaches have been utilized to attach novel side chain functionality into proteins in vivo in a site- (Chin et al., 2003a,b; Wang et al., 2001; Zhang et al., 2004), multisite- (Kwon et al., 2003), and residue-specific manner (Budisa et al., 1998, 2002; Kiick and Tirrell, 2000; Kiick et al., 2000, 2001; Tang and Tirrell, 2001; Tang et al., 2001a). The residue-specific incorporation of non-natural amino acids has been demonstrated by exploiting the aminoacyl- tRNA synthetases (AARS) (Budisa et al., 1998, 2002; Kiick and Tirrell, 2000; Kiick et al., 2000, 2001; Tang and Tirrell, 2001; Tang et al., 2001a). AARSs ensure the fidelity of amino acid incorporation into proteins by activating and charging the amino acid onto the tRNA (Ibba and Soll, 2000). Overexpression of wild-type (Kiick et al., 2000; Tang and Tirrell, 2001; Wang et al., 2003), mutant (Sharma et al., 2000), or heterologous (Furter, 1998) AARSs has expanded the ability of the translational apparatus to accept a variety of non-natural substrates. By utilizing an auxotrophic E. coli strain and feeding the cells with the non-natural amino acid, proteins bearing analogs in place of natural amino acids can be readily synthesized. Tirrell and coworkers have successfully incorporated the fluorinated amino acids trifluoroleucine (Tang et al., 2001a), hexafluoroleucine (Tang and Tirrell, 2001), trifluoroisoleu- cine (Wang et al., 2003), and trifluorovaline (Wang et al., 2004) with high levels of substitution into proteins in vivo. Flourinated amino acids are of particular interest due to their chemical inertness and increased hydrophobicity (Kukhar and Soloshonok, 1995). In particular, perfluorinated mole- cules such as trifluroromethyl groups are twice as hydro- phobic than methyl groups (Kukhar and Soloshonok, 1995; ß 2006 Wiley Periodicals, Inc. Correspondence to: J. K. Montclare Tatyana Panchenko and Wan Wen Zhu contributed equally to thework. Contract grant sponsors: National Institute of Health; Beckman Institute at the California Institute of Technology; Polytechnic University Start Up Funds Contract grant number: GM 62523, 5F32 GM67375-2