Lars Merkel

Azatryptophans endow proteins with intrinsic blue fluorescence.

Our long-term goal is the in vivo expression of intrinsically colored proteins without the need for further posttranslational modification or chemical functionalization by externally added reagents. Biocompatible (Aza)Indoles (Inds)/(Aza)Tryptophans (Trp) as optical probes represent almost ideal isosteric substitutes for natural Trp in cellular proteins. To overcome the limits of the traditionally used (7-Aza)Ind/(7-Aza)Trp, we substituted the single Trp residue in human annexin A5 (anxA5) by (4-Aza)Trp and (5-Aza)Trp in Trp-auxotrophic Escherichia coli cells. Both cells and proteins with these fluorophores possess intrinsic blue fluorescence detectable on routine UV irradiations. We identified (4-Aza)Ind as a superior optical probe due to its pronounced Stokes shift of ≈130 nm, its significantly higher quantum yield (QY) in aqueous buffers and its enhanced quenching resistance. Intracellular metabolic transformation of (4-Aza)Ind into (4-Aza)Trp coupled with high yield incorporation into proteins is the most straightforward method for the conversion of naturally colorless proteins and cells into their blue counterparts from amino acid precursors.

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

Highly fluorinated organic compounds exhibit a so-called “fluorous effect”, meaning that they are fluorophilic rather than hydrophilic or lipophilic. 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. 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. In the last decade, we and others demonstrated that single aromatic or aliphatic residues can be substituted by their fluorinated counterparts. However, in a proof-of-principle experiment, we very recently demonstrated incorporation of different noncanonical amino acids at the same time. 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 Cterminal 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, with great potential for industrial applications as well as synthetic biology. 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!(4F)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” in the UV absorbance spectrum of Tth-LipH6[(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 Scheme 1. Chemical structures of the canonical amino acids proline 1, 2, and 3 and their noncanonical counterparts 4, 5, and 6.

Engineering Protein Sequence Composition for Folding Robustness Renders Efficient Noncanonical Amino acid Incorporations

Although such experi-ments are usually performed with noncanonical isosteric ana-logues, such as monofluorinated amino acids, the introductionof more bulky moieties, for example, trifluorinated groups, canlead to drastic changes both in the surface properties of theprotein and the packing of its hydrophobic core, which in turnabolish the original biological activity.

Blue Fluorescent Amino Acids as In Vivo Building Blocks for Proteins

In vivo expression of colored proteins without post‐translational modification or chemical functionalization is highly desired for protein studies and cell biology. Cell‐permeable tryptophan analogues, such as azatryptophans, have proved to be almost ideal isosteric substitutes for natural tryptophan in cellular proteins. Their unique spectral features, such as markedly red‐shifted fluorescence, are transmitted into protein structures upon incorporation. Among the azaindoles under study (2‐, 4‐, 5‐, 6‐, and 7‐azaindole) 4‐azaindole has exhibited the largest Stokes shift (∼130 nm) in steady‐state fluorescence measurements. It is also highly biocompatible and as 4‐azatryptophan it can be translated into target protein sequences. However, its quantum yield and fluorescence intensity are still significantly lower when compared with natural indole/tryptophan. Since azatryptophans are hydrophilic, their presence in the hydrophobic core of proteins could be harmful. In order to overcome these limitations we have performed nitrogen methylation of azaindoles and generated mono‐ and dimethylated azaindoles. Some of these methyl derivatives retain the pronounced red shift present in the parent 4‐azaindole, but with much higher fluorescence intensity (reaching the level of indole/tryptophan). Therefore, the blue fluorescence of azaindole‐containing proteins could be further enhanced by the use of methylated analogues. Further substitution of any azaindole ring with either endo‐ or exocyclic nitrogen will not yield a spectral fluorescence maximum shift beyond 450 nm under steady‐state conditions in the physiological milieu. However, green fluorescence is a special feature of tautomeric species of azaindoles in various nonaqueous solvents. Thus, the design or evolution of the protein interior combined with the incorporation of these azaindoles might lead to the generation of specific chromophore microenvironments that facilitate tautomeric or protonated/deprotoned states associated with green fluorescence.

Fine tuning the N-terminal residue excision with methionine analogues.

Protein biosynthesis starts with methionine (Met) in all living cells. In the cytosol of eukaryotes and archaebacteria, protein translation is initiated with Met whereas N-formylmethionine (fMet) is used in eubacteria, the mitochondria and chloroplasts. The formyl group is added to the free a-amino group of Met-tRNA by the methionyl-tRNA formyltransferase (EC 2.1.2.9) and fMet-tRNA participates in translation. Met is always the first amino acid incorporated into the N-terminal position of a protein, even if alternative start codons, such as GUG and UUG are used for translation initiation. However, the amino acid sequence of mature proteins seldom contains Met at the first position; the N termini of most proteins are modified by a variety of co-, and post-translational processing events. Peptide deformylase (EC 3.5.1.88) removes the N-formyl group from proteins in prokaryotes and organelles. The unmasked amino terminal Met can subsequently be cleaved from prokaryotic as well as eukaryotic proteins by the Met aminopeptidase (MetAP; EC 3.4.11.18). Additionally, novel N termini are generated by the enzymatic cleavage of leader sequences, or by proteolytic digest at one or more positions within the polypeptide chain. The covalent attachment of diverse chemical functionalities, such as acetyl-, phosphate-, and myristoylgroups, or ubiquitin represents other N-terminal modifications beyond proteolytic processing. 13] As most N-terminal Met residues are coor post-translationally removed, their function lies in translation initiation rather than in structure. Depending on the organism, between 55 and 70 % of the proteins are subject to N-terminal Met excision (NME) by MetAP. NME is an irreversible cotranslational process that occurs as soon as the first N-terminal residues of the nascent peptide protrude from the ribosomal exit tunnel, before protein folding starts to occur. 15, 16] In eukaryotic cells, this enzymatic processing coincides with the intracellular localization of MetAP close to the ribosome. Although the localization of bacterial MetAP is still unclear (C. Klein, personal communication), it is reasonable to suppose that it is ribosome associated as well. MetAP specifically cleaves Met in the first position of the precursor protein. Meinnel and coworkers showed that model peptides with N-terminal Leu and Phe could be processed by Escherichia coli MetAP (EcMAP1) in vitro but with considerably lower catalytic efficiency than Met. The cleavage efficiency of Met in the first position largely depends on the bulkiness and nature of the side chain of the second amino acid (Figure 1). In vivo and in vitro data analyses provided convincing evidence that NME is likely to occur if the second residue of the precursor is Ala, Cys, Gly, Pro or Ser. Met in the first position can also be excised if Thr or Val follows in position 2, but with lower efficiency. Excision of the N-terminal Met with Ile and Asn at the second position is inefficient but may occur if the sequence context (vide infra) supports it. NME is not observed with the other amino acids at the second position. 13, 18] Although the data were collected predominantly in in vitro studies with EcMAP1 or in vivo with proteins from E. coli, MetAPs from diverse organisms all have the same specificity. 18] Taken together, smaller amino acids, especially Ala, at the second position optimally support N-terminal Met excision, whereas large side chains at the second position block the action of MetAP (Figure 1). According to earlier studies, the third position in the precursor protein does not influence NME. However, recently it was shown that not only the amino acid in position 2 but additional residues following the first Met have a major impact on NME efficiency. In vitro excision of the first Met from model peptides with either Ala or Gly in the second position was optimal if Trp, Met, or Ser followed in the third position. In conFigure 1. N-terminal Met excision (NME) is an irreversible cotranslational modification that is completed before the nascent polypeptide chain is fully synthesized. The basic rule is that small amino acid residues in the second position after the N-terminal Met facilitate NME, whereas bulky residues inhibit the process. MetAP, Met aminopeptidase.

In Vivo Chemoenzymatic Control of N-Terminal Processing in Recombinant Human Epidermal Growth Factor

Protein synthesis initiates with Met in the cytosol of eukaryotes and formylmethionine (fMet) in prokaryotes and eukaryotic organelles. N-terminal methionine excision (NME) is the major source of N-terminal amino acid diversity in all three life kingdoms. The excision is dictated by the nature and bulkiness of the side-chain of the second amino acid (the penultimate residue) and is catalyzed by methionylaminopeptidases (MetAPs; EC 3.4.11.18). In bacteria, Lys, Arg, Leu, Phe and Ile protect the N-terminal Met from removal, whereas Met excision is promoted by having Gly, Ala, Pro, Cys, Ser, Thr or Val as the penultimate residues (Scheme 1A). NME is an irreversible cotranslational proteolysis, and is completed before the nascent polypeptide chains are fully synthesized. It was recently estimated that between 55 and 70% of the proteins of any given proteome undergo this Met excision. The canonical amino acid, Met is recognized, activated and charged onto its cognate tRNAs by methionyl-tRNA synthetase (MetRS). The N-formyl group from the initiator, methionine is enzymatically removed from the nascent polypeptide in bacteria. This is an essential prerequisite for the co-translational action of MetAP. E. coli MetRS exhibits remarkable flexibility in its substrate binding and tRNA charging—a fact that we and others have used for in vivo incorporation of a large number of Met analogues and Met-like amino acids (surrogates) into polypeptide sequences. The chemical diversification of Met side-chains that can be achieved in this way is quite impressive; it ranges from aliphatic, chalcogen and halogen-containing side-chains, to unsaturated chemical groups like alkenes or alkynes and other interesting bioorthogonal groups such as azides. 10] Previously, Tirrell and co-workers demonstrated nearly quantitative substitution of the Met residues in dihydrofolate reduct ACHTUNGTRENNUNGase by homopropargylglycine (Hpg). The initiator Met was also substituted, but the Hpg in the first position was not excised. Similarly, we globally replaced Met with trifluoromethionine in green fluorescent protein and discovered an efficient blockage of the N-terminal excision of trifluoro-Met when Ser was the penultimate residue. Based on these observations we reasoned that the incorporation of different Met analogues at the protein’s N-terminus would enable us to change the NME rules in recombinant proteins. We chose to study the effects of azidohomoalanine (Aha) and Hpg (Scheme 1B), whose chemically unique azido and alkyne sidechains, respectively, have recently gained great importance for bioorthogonal transformations. In order to test the catalytic efficiency of MetAP towards these analogues, we prepared a series of pentapeptides that contain Met, Hpg or Aha as N-terminal amino acids with Arg or Gly in the second position (penultimate residues). While Arg, which is a bulky penultimate residue, was expected to efficiently block excision of the N-terminal residues, Gly is the smallest amino acid and should support NME. Expectedly, the overnight incubation of Arg2 pentapeptides (Met1-Arg2-Gln3-Leu4-Phe5; Aha1-Arg2-Gln3-Leu4-Phe5; and Hpg1-Arg2-Gln3-Leu4-Phe5) with recombinant E. coli MetAP yielded no cleavage of Met, Aha or Hpg from the peptides (data not shown). That means that the excision was blocked completely by having Arg in the second position. On the other hand, the excision of Met, Aha and Hpg was observed in the case of Gly2 pentapeptides (Met1-Gly2-Gln3-Leu4-Phe5; Aha1Gly2-Gln3-Leu4-Phe5; and Hpg1-Gly2-Gln3-Leu4-Phe5). The canonical amino acid Met was fully cleaved after 10 min, but complete excision of Aha and Hpg was observed only after 100 and 250 min, respectively (Figure 1). Although Aha and Hpg are less efficiently processed than Met in Gly2 pentapepACHTUNGTRENNUNGtides, they are indeed substrates for E. coli MetAP. Nonetheless, the main differences are found in the excision kinetics. These allowed us to identify the following order of the in vitro cleavage efficiency in Gly2 pentapeptides: Met>Aha>Hpg. Interestingly, the Aha-pentapeptide was better processed by the E. coli MetAP than the Hpg-pentapeptide, but we observed the opposite with MetAP from Pyrococcus furiosus (unpub[a] L. Merkel, Dr. Yu. Cheburkin, Dr. B. Wiltschi, Dr. N. Budisa Max Planck Institute of Biochemistry, Molecular Biotechnology Am Klopferspitz 18, Martinsried (Germany) Fax: (+49)89-8578-2815 E-mail : budisa@biochem.mpg.de Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. A) The structure of recombinant human epidermal growth factor (hEGF) with a marked N-terminus, along with the simple NME rules when Gly or Arg are the penultimate residues. B) Chemical structures of methionine (Met) and its analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg).

Convenient syntheses of homopropargylglycine

An improved classic Strecker synthesis was elaborated leading to racemic homopropargylglycine (Hpg) in 61% overall yield, while an asymmetric Strecker reaction produced Hpg and the higher homolog 2‐aminohept‐6‐ynoic acid in significantly higher yields and over 80% ee. Copyright

Synthetic Biology of Autofluorescent Proteins

Abstract Autofluorescent proteins (FPs), which to date are predominately used as tools in cell biology and spectroscopy, have arrived in the focus of synthetic biology. Thereby, the intention is to supplement classically used protein design methods such as site-directed mutagenesis or guided evolution by expanding the scope of protein synthesis. This is achieved by the co-translational introduction of novel noncanonical amino acids (NCAAs) into proteins. In the following chapter, we present current applications of an expanded amino acid repertoire for the design of spectral and folding properties of FPs. We will show that NCAAs are not only useful tools to study fundamental aspects of photophysics but also have great potential to generate novel FP tools for cell biology applications. On the one hand, aromatic amino acids other than the naturally occurring His, Tyr, Phe, and Trp were used to create novel spectral classes of FPs by direct chromophore modification. On the other hand, NCAAs were also applied for “FP protein matrix engineering” to influence chromophore fluorescence and overall folding. We also illustrate a practical application of these principles by presenting “golden annexin A5” as a novel apoptosis detection tool designed by synthetic biology methods. Finally, we describe a potential route to convert any protein of interest into a chromo-protein by introduction of novel synthetic autofluorescent amino acids.