Protein Design Using Unnatural Amino Acids

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Products of Chemistry

George B. Kauffman California State University Fresno, CA 93740

Protein Design Using Unnatural Amino Acids Basar Bilgiçer and Krishna Kumar* Department of Chemistry, Tufts University, Medford, MA 02155; *[email protected]

In 1958, the first crystal structure of a globular protein, myoglobin, was determined by John Kendrew (1, 2). In a radical departure from the remarkable simplicity of the double-stranded DNA structure solved just five years earlier by X-ray diffraction analysis (3), the structure of myoglobin was complex and presented an apparent lack of symmetry coming as a surprise to those who had expected simple and global rules for protein structure. As later structures would reveal, structural complexity is a necessary aspect for the wide variety of functional tasks that proteins undertake. In the decades to follow, researchers probed the function of proteins using a variety of techniques, but were limited to the sequences and structures that existed in nature. As recombinant DNA techniques became sophisticated enough to allow specific genetic manipulation, the first report of oligonucleotide-directed mutagenesis to study protein function appeared in 1982, and heralded a new era in enzymology and biological chemistry (4, 5). Site-directed mutagenesis allowed the replacement of any amino acid in the primary sequence of a protein by one of the twenty natural amino acids. This powerful method has been used extensively to probe biochemical and biophysical properties of ‘mutants’, and by comparison to the ‘native’ enzyme, has often lead to key insights into understanding structure, function, and the mechanism of catalysis. Conventional site-directed mutagenesis, while far reaching in its impact and utility, is still limited to the 20 side chains of the proteinogenic amino acids. For a long time, chemists have had a large arsenal of unnatural alpha amino acids that could, in principle, provide more detailed and relevant information about the structure and function of proteins. However, only recently has it been possible to introduce noncoded amino acids site-specifically using both chemical synthesis or biosynthesis. In just a little more than ten years, more than 100 different amino acids have been site-specifically incorporated by biosynthetic methods (6). A fair number of review articles have described the successes and limitations of the various approaches (6, 7). Bearing this in mind, our focus is on two aspects, first to provide an overview of existing techniques of incorporation and their limitations, and second, to highlight recent examples in which the introduction of unnatural amino acids into model proteins has resulted in dramatic enhancements in the stability of the folded forms. The intent is to highlight a new direction for protein and peptide design. Methods of Incorporation The oldest method of biosynthetic incorporation of unnatural amino acids is the use of auxotrophs—bacterial strains

that are unable to synthesize one or more amino acids (8– 11). An unnatural amino acid analog that is to be incorporated is supplied to the auxotroph during growth, resulting in its incorporation in the expressed proteins. There are several limitations to this method. First, site-specific incorporation is not possible. Second, if proteins have more than one residue corresponding to the unnatural analog supplied, then there is virtually no control over the number of amino acids that will be substituted. Indeed, heterogeneous mixtures are commonly produced; that is, substitution fails at one or more positions. Third, the method is limited to structural analogs of the knocked-out amino acid. Some of these problems are circumvented by the use of in vitro translation in cell-free extracts. The advantage is clearly that analogs of any of the natural amino acids may be incorporated. Furthermore, the ratio of the unnatural to the natural amino acid can be accurately controlled. However, the efficiency of this method is far lower than the in vivo approach. The second method of biosynthetic incorporation of unnatural amino acids involves the use of naturally occurring suppression of nonsense (stop) codons. In the most widely used version of this approach, the codon for the amino acid residue to be substituted is replaced with an amber termination codon (TAG) in the gene for the desired protein (12– 15). The gene is then incorporated in a plasmid under the regulation of an inducible promoter. The complementary anticodon containing tRNA is then charged by semisynthesis to carry the unnatural amino acid (16). These components then typically form part of a cell-free expression system that results in the production of the suitably modified mutant protein (Figure 1). There have been reports of improvements to this basic scheme to obtain higher yields of protein and also to cut down the number of steps required. Higher yields are critically important as even the best cases have resulted in quantities approaching only 1 mg of protein. Other improvements have also been made to this technology. Notable among these is the work of Lester, Dougherty, and coworkers, who have extended this method to achieve nonsense suppression in vivo in Xenopus oocytes (17, 19). They have also developed the use of tRNAGln,CUA from Tetrahymena thermophila in the oocyte system. Kowal and Oliver have used the eubacterium Micrococcus luteus as an alternative suppression system (20). Because the eubacterium purportedly cannot translate as many as six codons, this opens up the possibility of simultaneously introducing more than one type of unnatural amino acid and is likely to work at positions where codons cannot be suppressed readily. Recently, Schultz and coworkers reported a modified stop-codon suppression method (21). Both the aminoacyl tRNA synthetase and the tRNA genes of an

JChemEd.chem.wisc.edu • Vol. 80 No. 11 November 2003 • Journal of Chemical Education

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Figure 1. In vitro site-directed mutagenesis to incorporate unnatural amino acids. Site-directed mutagenesis is used to change a specific codon to an amber termination codon (TAG). The plasmid is then part of an in vitro expression system that contains the appropriate aminoacyl suppressor tRNA. The end result is the production of a protein with an unnatural amino acid (shown in space filling depiction) in response to the termination codon.

archaebacterium were inserted into E. coli. The tRNA was modified so that it recognized the stop codon and the synthetase was modified using protein evolution methods to catalyze the addition O-methyl-L-tyrosine to the tRNA. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-L-tyrosine into proteins in response to an amber nonsense codon. The technical advance here is that synthesis of the aminoacylated tRNA is obviated. However, a new pair of anticodon modified tRNA and a modified synthetase that catalyzes the addition of the unnatural amino acid needs to be developed for every new amino acid. The most direct method of introducing unnatural amino acids is of course through chemical synthesis. In practice however, peptides larger than 20 residues were not easily synthesized until about a decade ago. Nowadays, synthesis of 50 residue peptides must be considered routine and through two important advances in peptide ligation technology, proteins with up to 200 residues may be prepared (Figure 2). The first of these is native chemical ligation, which allows the preparation of small proteins with native backbone structures from fully unprotected synthetic peptides under mild aqueous conditions (22). In this approach, a peptide containing a Cterminal thioester is reacted with a N-terminal cysteinecontaining peptide. First, the N-terminal cysteine sulfhydryl reacts with the thioester in a transesterification reaction. Second, an irreversible intramolecular S to N acyl-transfer reaction takes place resulting in the native amide bond. One limitation of this method is the size of the C-terminal thioester fragments that may be prepared by solid-phase synthesis. To solve this problem, Muir and Xu have independently introduced expressed protein ligation, a method in which the C-terminal thioester fragment is prepared biosynthetically by taking advantage of protein splicing, a naturally occurring process that involves thioester intermediates (23, 24). This strategy allows synthetic peptides to be attached to large recombinant proteins and allows the simultaneous inclusion of many unnatural amino acids. Both these methods suffer 1276

from the mandatory use of N-terminal cysteine residues in one of the coupling partners, although Dawson and coworkers have recently reported successful desulfurization after ligation (25). In spite of some limitations, the technology is very powerful and has changed the landscape of the protein sizes attainable by synthesis. Furthermore, incorporation utilizing synthesis offers variations in stereochemistry (D-amino acids) and in backbone diversity (e.g., β-amino acids) that traditional biosynthetic systems do not. Applications The methods summarized above have resulted in the incorporation of more than 100 unnatural amino acids into proteins. These studies have revealed the intricate details of the basis for protein stability, protein–membrane interactions, enzyme–substrate specificity, and protein–protein interactions. Furthermore, photoactivable side chains, biophysical and structure-function probes have also been introduced. There now exists a quorum of knowledge that would not have been possible without the extra-biological side chains of unnatural amino acid residues. Caged Side Chains The most common form of caging has been the use of o-nitrobenzyl group attached to a heteroatom present on the side chain (26). Upon irradiation, the protecting group is removed revealing hidden functionality. Schultz and coworkers incorporated a caged serine that was photochemically deprotected and initiated protein splicing (27). While it was already clear that the particular serine was catalytically important, this study demonstrated how a switch could be engineered to initiate biochemical events. Schultz and coworkers have also used the o-nitrobenzyl ester of aspartic acid to control the protein–protein interface between the p21ras (ras) and the p120–GAP (28). Dougherty, Lester, and coworkers have

Journal of Chemical Education • Vol. 80 No. 11 November 2003 • JChemEd.chem.wisc.edu

In the Classroom native chemical ligation

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Figure 2. Native chemical ligation (left) and expressed protein ligation (EPL, right). The key step in native chemical ligation is the S to N acyl transfer resulting in a native amide bond. The advance in EPL is the formation of the thioester in a N to S acyl transfer step during protein splicing, which is then followed by native chemical ligation.

incorporated 2-(nitrophenyl)glycine into ion channel proteins in vivo for mechanistic studies. Upon irradiation, the protein is cleaved specifically at the site of the unnatural amino acid in a manner similar to proteolysis (29). They have also used a caged tyrosine to look at time-resolved events at the agonist binding site of the nicotinic acetylcholine receptor (nAChR). The receptor was rendered nonfunctional presumably owing to the physical bulk of the protecting group on the tyrosine hydroxyl, but activity was restored upon deprotection (30). Caged side chains provide a trigger to control the side chain behavior of proteins and may be useful in elucidating signal transduction pathways.

Biophysical Reporter Groups Several studies detailing the incorporation of fluorescent groups have been reported (31–33). Schultz and coworkers have shown that a side chain nitroxyl group could be successfully incorporated serving as a spin label (34). Residuelevel information is essential to understanding important questions about protein folding, structure, and function. Most common techniques however usually only give a global view of structure. NMR is capable of providing residue level information but gets very complicated for large proteins when uniform isotopic labeling is utilized. The use of site-


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specific unnatural side chains or isotopically-substituted main chain atoms can provide residue-specific structural information. Perhaps the most promising of these is the segmental isotopic labeling strategy (35). In this approach, segments of entire proteins are isotopically backbone (or side chain) labeled with NMR active nuclei. Large proteins have an insurmountable redundancy in chemical shifts complicating solution structure determination. This method is likely to increase the size of proteins that can be studied with NMR methods. Muir and Cowburn have used this technology to assemble the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with N15. Recently, the 304 amino acid eukaryotic adaptor protein, Crk-II, was assembled from three recombinant polypeptide segments in good yield (36–37). These reports are encouraging and suggest that internal regions of large proteins are now accessible to labeling with NMR active nuclei. Engineered Ester Backbones

and Y are often L-proline (Pro) and 4(R)-hydroxyl-L-proline (Hyp), respectively. It was well known that Hyp residues dramatically increase the thermal stability of triple-helical collagen. However, the reasons for this increased stability were ambiguous. Structural models based on experimental data suggested that main chain hydrogen bonding involving the hydroxyl at the 4-position was not possible. Soon, models invoking hydrogen bonding with bridging water molecules and the 4-hydroxyl were put forward. Raines and coworkers argued that already existing evidence pointed to a different mechanism of stabilization. In a series of elegant studies, they have now unambiguously established the mechanism by which the hydroxyl at the 4-position increases the overall stability of the collagen triple-helix (44–47). They synthesized a collagen-like peptide with Pro-Flp-Gly repeats, where Flp is 4(R)-fluoro-L-proline. The resulting assembly had all the characteristics of triple-helical collagen but was hyperstable. Indeed, the highly electronegative fluorine atom in the 4(R) position conferred a ∼20 ⬚C increase in thermal stability and each fluorine atom contributed roughly 0.2 kcal兾mol in

There has been interest in probing the effect of ester linkages introduced in the backbone to evaluate the strength of hydrogen bonding interactions on the stability of proteins. Schultz and coworkers have introduced ester linkages by use of α-hydroxy acids in T4 lysozyme (38) and staphylococcal nuclease (39). They found that depending on the location of the ester linkages, the protein was destabilized by 0.7–2.5 kcal兾mol. The ester carbonyl is similar in structure to the amide but is a weaker hydrogen bond acceptor, and the hydrogen bond donating NH is replaced with the electronegative oxygen atom of the ester. Using total synthesis, Beligere and Dawson synthesized chymotrypsin inhibitor 2 (CI2), a small 64 residue protein consisting of an α-helix sandwiched by four strands (40). A total of four amide bonds that span the length of the α-helix were replaced with ester bonds. The resulting 4-ester CI2 was a functional protease inhibitor but was less stable to chemical denaturation by 2.93 kcal兾mol. Esters have also been introduced throughout the transmembrane region of nAChR. The study supports a gating model that includes changes of backbone conformation within the M2 domain of the receptor (41). Fluorinated Amino Acids in the Design of Stable Folds Biological molecules do not usually contain the element fluorine. However, the properties of fluorine make it an invaluable element for design and synthesis of novel protein folds and biophysical probes. There has been a recent surge of interest in using fluorinated amino acids in the design of hyperstable peptides and proteins. While the use of fluorinated amino acids as reporter groups has been reported in many studies earlier (42–43), recent advances have utilized the unique properties of the fluorinated amino acids to design stable protein motifs. Three of these studies are described in detail below. Hyperstable Collagen Mimics Collagen is a fibrous protein found in animals that occurs in various different forms. There are 300 repeats of the sequence Gly-X-Y in a typical polypeptide segment where X 1278

Figure 3. (Left) Electronegative substituents at the 4(R) position in hydroxyproline or fluoroproline (OH or F) favor the trans amide bond. Model compounds exhibit this trend shifting the equilibrium toward the trans isomer. (Right) Since triple helical collagen contains only trans amide bonds (48), substituents at this position exert a powerful stereoelectronic effect in stabilizing the three-dimensional structure of collagen.


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∆∆Gm over the hydroxyproline structure. The authors have noted that both 4(R)-hydroxy-L-proline and Flp stabilize the trans configuration of the amide bond. Since the amide bond is trans in triple-helical collagen, the highly electronegative 4(R) substituent enhances conformational stability by favoring the trans isomer by pre-organization of individual strands to mimic their conformation in the final structure (Figure 3). Fluorinated Coiled Coils The coiled coil domain is a widespread structural pattern found in fibrous as well as globular proteins (49). Nearly 5% of all open reading frames contain coiled coils in genomes that have been sequenced (50). These structures are very amenable to protein design studies, as the interactions governing structure are well understood. Furthermore, they are one of the very few protein motifs that have yielded predictably to computation. Coiled coils self-assemble into oligomers: the primary driving force being hydrophobic interactions. The sequences exhibit a 4–3 hydrophobic repeat (abcdefg)n, with

Figure 4. (A) A schematic view of coiled coils: Left–looking down the helical axis and the heptad repeat. The hydrophobic core is shown green. Right–two helices pack together to give a dimer. (B) Computer generated image of a 32-residue coiled coil protein containing two helices. The core in the native protein is hydrocarbon1 and in the hyperstable designed version shown here is hydrocarbon-2, all core leucine (L) and valine (V) residues have been replaced by trifluoroleucine (L) and trifluorovaline (V) residues respectively. The asterisk indicates a secondary stereocenter in L and V. A total of 14 trifluoromethyl groups are buried in the hydrophobic core. (Color scheme: gray—carbon, red—oxygen, blue—nitrogen, white—hydrogen, yellow—sulfur, and green–fluorine.)

the a and d positions predominantly occupied by amino acid residues with hydrophobic side chains. The a and d residues fall on one face of the helix providing a nice surface for intermolecular interactions (Figure 4). Charged residues are commonly found at the e and g positions. Side chains of the residues at these four positions participate in interhelical hydrophobic and electrostatic interactions. The solvent-exposed positions b, c, and f are not directly involved in the self-assembly process and can tolerate a wide variety of amino acid substitutions. We, and others, have recently described the design and synthesis of coiled coils with highly fluorinated cores. Tirrell and coworkers have successfully replaced all four hydrophobic residues at the d position of GCN4-p1 with trifluoroleucine, the coiled coil region of a eukaryotic transcription factor, using both in vivo and synthetic methods (51, 52). They argued that trifluoroleucine might behave as a hyper-hydrophobic analogue of leucine. Indeed, using circular dichroism, they found that the fluorinated GCN4 exhibited both enhanced thermal stability (∆Tm = 13 ⬚C) and was more resistant to denaturation by chemical agents. Our laboratory has synthesized fluorinated coiled coil proteins based on GCN4-p1 by replacing all four leucine residues (d position) and three valine (a position) with trifluoroleucine and trifluorovaline, respectively (Figure 4; ref 53). The fluorinated peptide was highly α-helical at low micromolar concentrations as judged by circular dichroism spectra, sedimented as a dimeric species and exhibited a dimer melting temperature that was 15 ⬚C higher than a control peptide with a hydrocarbon core. Furthermore, the apparent free energy of unfolding as calculated from guanidine hydrochloride denaturation experiments was larger by ∼1.0 kcal兾mol for the fluorinated peptide than its hydrocarbon counterpart. Additional stability of the fluorinated peptides is derived from the higher hydrophobicity of the fluorinated residues that are removed from solvent upon dimerization. Simple perfluorocarbons are similar to hydrocarbons in that they are hydrophobic; however, owing to the low polarizability of fluorine, they have very low propensity for intermolecular interactions, quite unlike hydrocarbons. To avoid loss of favorable van der Waals interactions within the hydrocarbon phase, mixtures of perfluorocarbons and hydrocarbons remain phase separated at room temperature. We have recently used the stability of the fluorinated proteins to direct the self-sorting of peptides that have either hydrocarbon (H), fluorocarbon (F), or a mixed hydrocarbon– fluorocarbon core (54, 55). The preference for sorting into homodimeric populations under equilibrium conditions was examined by a disulfide-exchange assay (Figure 5). Three peptide assemblies, each containing two peptide fragments coupled at their N-termini by a disulfide linkage were synthesized. The cores formed by these peptides were composed of exclusively hydrocarbon (HH), fluorocarbon (FF) or a combination of both H and F helices (HF). Preformed disulfide bonded heterodimer HF was incubated in a pH 7.50 redox buffer at 20 ⬚C, conditions under which disulfide exchange is rapid. Aliquots were removed from the reaction at various times and quenched with 5% trifluoroacetic acid. Within 30 min of the start of the reaction, the heterodimer disproportionates into the two homodimers HH and FF. After 200 min, only a trace of the heterodimer remains in solution. Further change in the reaction mixture was not


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Literature Cited

Figure 5. Self-sorting in a mixture of N-terminal cysteine containing peptides HH (hydrocarbon core) and FF (fluorous core). Preformed heterodimeric disulfide bridged compound HF is allowed to equilibrate under redox buffer conditions (disulfide exchange is rapid). The heterodimer sorts almost entirely into the homodimeric species HH and FF. The fluorinated and hydrocarbon peptide prefer to make homodimers owing to the relative instability of the heterodimer.

observed even after 18 h. Assuming that the glycyl linkers allow the cysteines to exchange randomly under redox buffer conditions, the data indicate that the homodimers are preferred over the heterodimer. Biophysical studies established that the relative instability of the heterodimer and the hyperstability of the fluorinated dimer provide the driving force (∆Gspec = ᎑2.1 kcal兾mol) for preferential homodimer formation. The fluorinated residues provide an easy and novel means to increase the conformational stability of the proteins. With many methods now available to incorporate unnatural amino acids into proteins, it is only a matter of time before a large number of proteins containing fluorinated residues are designed and synthesized. Outlook Recent advances in methods of incorporation of unnatural amino acids into proteins have made it possible to choose from an unlimited arsenal of side chains. The full gamut of side chain architectures will soon be available and will lead to new protein folds and novel function. These efforts will undoubtedly usher in a new era in both protein design and in the exploration of protein function. With increasing availability of whole organism genome sequences, a fundamental understanding of protein structure and function takes on added importance. Coupled with the more ambitious efforts being directed at the expansion of the genetic code for programmed incorporation of unnatural amino acids (21, 56), the face of protein chemistry and biology is certain to be changed forever. Acknowledgments We thank the National Institutes of Health (GM65500) and the National Science Foundation (CHE-0236846) for support. We also thank Marc d’Alarcao for stimulating discussions. 1280

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