Parallel Incorporation of Different Fluorinated Amino Acids: On the Way to “Teflon” Proteins (2010)

Nonstick proteins? We report the successful residue-specific fluorination of 24 positions in a lipase by simultaneous incorporation of three different noncanonical amino acids. The enzyme produced in a polyauxotrophic E. coli strain structurally tolerates all fluorine atoms and retains its catalytic activity very well. Highly fluorinated organic compounds exhibit a so-called “fluorous effect”, meaning that they are fluorophilic rather than hydrophilic or lipophilic.1, 2 This property might be transmitted to enzymes by endowing them with novel and special features and functions such as solvent resistance, conformational stability, enhanced activity, lipophilicity, shifted temperature, or pH optima. In order to produce such protein variants in good yields, supplement incorporation (SPI) is certainly the method of choice.3 It enables highly efficient and simple residue-specific fluorination of amino acids in different recombinant proteins. In an ideal case, isosteric fluorinated analogues can be simultaneously incorporated in a single protein in just one expression experiment, without affecting its structure.3 In the last decade, we and others demonstrated that single aromatic or aliphatic residues can be substituted by their fluorinated counterparts.4–9 However, in a proof-of-principle experiment, we very recently demonstrated incorporation of different noncanonical amino acids at the same time.10 In the next step, which is presented here, we aimed to substitute three amino acid residues simultaneously with their monofluorinated analogues in a single expression experiment. In particular, all proline (Pro, 1), phenylalanine (Phe, 2), and tryptophan (Trp, 3) residues (Scheme 1) should be substituted by their noncanonical counterparts 4(S)-fluoroproline ((4(S)-F)Pro, 4) 4-fluorophenylalanine ((4-F)Phe, 5), and 6-fluorotryptophan ((6-F)Trp, 6) in a single enzyme. The highly relevant industrial enzyme lipase from Thermoanaerobacter thermohydrosulfuricus was chosen for this experimental attempt. After fusion to a C-terminal His6 tag (Tth-Lip-H6), it consists of 267 amino acids that harbor six proline, 16 phenylalanine, and two tryptophan residues (see the Supporting Information). We intended to investigate to what extent such multiple residue-specific global substitutions, which include approximately 10 % of the primary sequence, will be tolerated by the protein structural plasticity. The other fundamental question is whether structural integrity and enzymatic activity will be preserved upon this high degree of global fluorination. This would certainly represent an important step toward the generation of highly fluorinated proteins as a type of “Teflon” protein,1 with great potential for industrial applications as well as synthetic biology. Chemical structures of the canonical amino acids proline 1, 2, and 3 and their noncanonical counterparts 4, 5, and 6. After expression (see the Experimental Section), cell harvest, and subsequent lysis, NiNTA purification was performed, resulting in pure protein variants in good yields. However, the massive sequence modification lowered the yield of the multifluorinated variant Tth-Lip-H6[(4(S)-F)Pro]/[(4-F)Phe]/[(6-F)Trp] to 11.5 mg L−1 culture compared with a 47 mg L−1 culture for the parent Tth-Lip-H6. Nonetheless, we could obtain sufficient material to perform the required analytical examinations. The mass spectrometric analyses (ESIMS) clearly revealed a high level of the target compound without significant traces of the parent protein, confirming full substitution of all residues of interest in the lipase, that is, 6 Pro→(4(S)-F)Pro; 16 Phe→(4-F)Phe; 2 Trp→(6-F)Trp. Although the signal corresponding to the fully labeled Tth-Lip-H6[(4(S)-F)Pro]/[(4-F)Phe]/[(6-F)Trp] was the highest intensity signal in the ESIMS profile, signals corresponding to species with only 23 and 22 fluorinated amino acids were also detectable. In order to analyze the occupancy of fluorine at each modified site in the sequence, in-gel digestion was performed, and the resulting tryptic fragments were analyzed by ESI–MS and MS/MS. As expected, very high fluorine occupancy at all sites was observed. This clearly indicates that fluorination was uniformly distributed among all substitution sites of the lipase sequence in a statistical manner. The trace presence of canonical amino acids could be explained by intracellular “leakage”, which is most probably due to the turnover of the canonical amino acids in the polyauxotrophic E. coli strain ME5355. Interestingly, the high level of substitution by the fluorinated noncanonical amino acids is also mirrored in the spectroscopic characteristics of the Tth-Lip-H6 variant. For example, the incorporation of (4-F)Phe results in the appearance of so-called “fluoro fingers”11 in the UV absorbance spectrum of Tth-Lip-H6[(4(S)-F)Pro]/[(4-F)Phe]/[(6-F)Trp] (Figure 1 A). The fluorescence emission spectrum is only slightly affected by the incorporation of (6-F)Trp as shown in Figure 1 B. Namely, the slight red shift of 13 nm (from 331 to 344 nm) is associated with the presence of (6-F)Trp in the lipase structure as previously observed in an analogous annexin A5 variant.12 Comparison of the spectral and enzymatic properties of the parent lipase (—) and the fluorinated variant Tth-Lip-H6[(4(S)-F)Pro]/[(4-F)Phe]/[(6-F)Trp] (••••). A) UV absorption spectra (note the appearance of fluoro fingers at 261 and 267 nm in the fluorinated lipase). B) Fluorescence emission profiles upon excitation at 280 nm (NF: normalized fluorescence). C) Far-UV CD spectra of parent and fluorinated lipase at 20 °C. D) Specific activity of both proteins. For experimental details see the Supporting Information. The secondary structure of Tth-Lip-H6 is not significantly affected by incorporation of fluorinated noncanonical amino acids as indicated by circular dichromism (CD) spectroscopy (Figure 1 C). The slight increase in the dichroic minimum at approximately 208 nm could suggest local perturbations in the structure at low extents, a fact that can be examined only by high-resolution crystallographic studies. In the near-UV spectral region, CD analysis indicates significant spectral differences between the parent protein Tth-Lip-H6 and its fluorinated variant (see the Supporting Information). This is not surprising because 16 Phe and two Trp residues in the lipase structure are fluorinated, a fact that is expected to affect the “aromatic” region in the near-UV CD spectra. In general, all three exchanged residues, Pro, Phe, and Trp, play an important role in the lipase activity. For example, Phe residues are located in the binding cavity in all lipases and are expected to influence the enzyme kinetics. The enzymatic activity was determined with a standard photometric assay by using p-nitrophenyl palmitate as a chromogenic substrate and monitoring the released p-nitrophenol by UV spectroscopy at 410 nm.13 Although Tth-Lip-H6 exhibits its highest efficiency at 70 °C, the maximum activity of the fluorinated variant occurs at 60 °C. However, this maximum is only about 60 % that of the parent protein (Figure 1 D). Nonetheless, the enzyme activity is not critically affected when the large-scale sequence fluorinations are taken into account. On the other hand, the decrease of Topt of a lipase could well represent a highly desired feature, for example, as in the industrially relevant design of “cold-wash” lipases. Finally, the effects of highly fluorinated amino acids or analogues with other ring substituents in different aromatic amino acids, as well as their combinations, might yield completely different and unexpected features. We are currently investigating these phenomena, in combination with different substrates, solvents, and experimental conditions. To the best of our knowledge, this is the first successful incorporation of three different monofluorinated amino acids at 24 positions in a single protein in one expression experiment. Most importantly, these large sequence modifications (∼10 % residues) did not induce detrimental structural effects or abolish lipase enzymatic activity. We are well aware that, in most cases, noncanonical analogues such as monofluorinated amino acids are better tolerated than bulky ones, for example, trifluorinated moieties. The incorporation of groups with high spatial demands can lead to drastic changes both in the surface properties of the protein and the packing of its hydrophobic core, abolishing the original biological activity of the parent molecule. One way to circumvent these problems is to perform such fluorinations in combination with guided enzyme evolution. In this way, target protein compositions that are highly amenable for such replacements can be identified as demonstrated recently.14 We intend to combine this strategy with our multiple-incorporations approach, which includes various perfluroinated amino acids. We are confident that this is the correct strategy to generate not only “Teflon” proteins in the future, but also to produce novel properties in proteins that have never been encountered in the natural biology realm. E. coli strain ME5355 (thi pro arg trp tyr phe aroB) served as the expression host for in vivo parallel incorporation experiments with multiple-site and multiple-type fluorinated amino acids. Although this strain requires the addition of shikimic acid (cfinal=10 μm) when cultured in minimal media, it proved to be a very efficient expression platform (see below). After determining the limiting concentrations (for OD600∼0.8: 1=50, 2=40, 3=7.3 μM), cells were grown in new minimal medium (NMM)15 that was supplemented with the required amounts of 1, 2, and 3. The depletion of the canonical amino acids is indicated by a growth arrest. After reaching a constant OD600 level, a media shift by using NMM without 1, 2, and 3 was performed, and the cells were incubated again for 1 h at 37 °C. Subsequently, the noncanonical amino acids 4, 5, and 6 were added (cfinal=0.5 mM), the expression of the Tth-Lip-H6 variant was induced with IPTG (cfinal=1 mM), and the cells were incubated for 4 h at 30 °C. In addition, expression of Tth-Lip-H6 with 1, 2, and 3 was performed as a reference under the same conditions. Elisabeth Weyher-Stingl and Dr. Cyril Boulegue are gratefully acknowledged for their excellent assistance in analytics. The National BioResource Project, Japan, is greatly acknowledged for providing bacterial strains. This work was supported by the BioFuture Program of the Federal Ministry of Education and Research of Germany. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. 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