Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Aminotryptophan-containing barstar: Structure–function tradeoff in protein design and engineering with an expanded genetic code
Introduction
One notable property of protein translation that did not escape the attention of many researchers is that it is performed with an invariant set of 20 amino acids prescribed by the universal genetic code [1]. The chemical diversity encoded in this way includes a defined number of side chain functional groups: carboxylic acids (Glu, Asp), amides (Gln, Asn), thiol (Cys) and thiolether (Met), alcohols (Ser, Thr), amines (Lys), guanidine (Arg), aliphatic (Ala, Val, Leu, Ile) and aromatic side chains (Phe, Tyr, His, Trp), and even a cyclic imino acid (Pro). On the other hand, a remarkable tolerance of the ribosome (i.e., ribosome plasticity) towards many amino acid types ranging from derivatives of biotin to polycyclic aromatic systems is now well documented [2]. A vast number of amino acids can be translated into protein sequences. Examples include amino acids with increased side chain bulk, novel chemical groups and functionalities (e.g., amino, cyano, nitro, nitroso, keto, azido, alkene, alkyne) and atoms such as various halogens or chalcogens and even novel backbone chemistries [3], [4], [5], [6]. Obviously, ribosome-mediated synthesis of proteins provides a sufficient room for increasing the chemical diversity of the genetic code vocabulary. Proteins generated in this way often possesses unique properties transcending those found in nature and might serve as useful tool for understanding the precise molecular basis of protein structure–function relationships, ligand binding, conformational change and protein–protein interactions [7].
The expansion of the amino acid repertoire in protein biosynthesis is possible since numerous noncanonical analogs and surrogates of canonical amino acids can act as substrates for translation machinery. This requires their intracellular accumulation by the cellular uptake systems. These systems and the aminoacyl-tRNA synthetases (AARS), crucial enzymes in the interpretation of the genetic code, often cannot distinguish between structurally or chemically similar amino acids [8]. In the experimental approach termed as selective pressure incorporation (SPI), these features are exploited. The basic principle of this method is that the choice of amino acids for protein synthesis can be conditioned by the control of environmental factors by changing the fermentation parameters. In this way, a controlled expression of a target protein in bacterial cells which are auxotrophic for particular amino acids is accompanied by ‘mis-incorporations’ of the related analog. Such a reprogrammed translation yields proteins with a changed chemical composition. In summary, SPI is now a well-established methodology that essentially requires the availability of metabolically engineered cells (auxotrophs) as expression hosts and controllable expression conditions [9]. This provides a basis for efficient amino acid replacement by codon reassignment on the level of a single protein [10].
The canonical amino acid Trp possesses at least three salient features: (i) low natural abundance in proteins (only ∼ 1%), (ii) unique biophysical and chemical properties, and (iii) its involvement in a variety of biological interactions and functions [11]. In addition, Trp encoded by a single UGG triplet is the main reporter of intrinsic fluorescence in most proteins and peptides. Thus, it is an attractive target for tailoring the spectral properties of proteins. In our previous studies, it has been shown that the spectral function of proteins can be engineered by the incorporation of various hydroxy-, amino-, halogen-, and chalcogen-containing Trp analogs and surrogates [12].
Aminotryptophans have a special place among all these substances. From a chemical point of view, they represent a class of aromatic amines that are much weaker bases than aliphatic amines. Further, (4-NH2)Trp and (5-NH2)Trp are also interesting due to their structural similarity to the natural DNA base adenine. In adenine, the exocyclic aromatic amino group is often a key recognition element in the formation of nucleic acid structures and in intermolecular interactions of DNA/RNA and proteins. In addition, the fluorescence properties of all four DNA bases are characterized by the existence of an excited state with charge transfer character [13]. During translation with (4-NH2)Trp and (5-NH2)Trp, these properties can be transmitted into the resulting protein structure as a response to all UGG triplets present in target sequence. Proteins synthesized in this way are endowed with a pH-sensitive fluorescence which results from charge transfer [14]. In other words, a variation in solvent pH causes a predictable change in the optical properties of these protein variants which might be an interesting practical application. In classical chemistry, aromatic amines are basic substrates for the synthesis of new pigments and dyes. Similarly, the incorporation of aminotryptophans into the “cyan” mutant of the green fluorescent protein (GFP) has yielded a protein with a novel chromophore. This specific engineering approach has given rise to a unique “gold” class of autofluorescent proteins with unique chromophore photophysics [15].
However, an important issue to be answered is about the thermodynamic price that must be paid in order to generate such proteins with tailored functions. Although rarely appreciated in current studies [16], this issue is highly relevant for protein design and engineering with an expanded genetic code. In order to address this problem, we extended our previous spectroscopic studies [14] on barstar as model protein by additional thermodynamic, X-ray and modeling investigations of (4-NH2)Trp- and (5-NH2)Trp-barstar. To dissect the influence of the amino group on the determined thermodynamic features of these protein variants, the incorporation of methylated Trp analogs, like 4-, 5-, 6-, and 7-methyltryptophan ((4-Me)Trp, (5-Me)Trp, (6-Me)Trp and (7-Me)Trp), into barstar has been attempted as well (Fig. 1). Indeed, the tryptophan → aminotryptophan → methyltryptophan replacement is a model for a –H → –NH2 → –CH3 isosteric substitution in proteins. Experiments reported in this study are expected to provide important insights into the fundamental roles of aromatic amines in structural stability and functions of tailor made proteins synthesized by the ribosome machinery.
Section snippets
Chemicals, expression host, fermentation, protein labeling, expression and purification
The noncanonical Trp-analogs (4-NH2)Trp and (5-NH2)Trp were synthesized enzymatically as described elsewhere [14]. All other chemicals were purchased from Sigma-Aldrich unless stated otherwise. Tryptophan auxotrophic bacterial expression host E. coli ATCC49980 (genotype: WP2 uvrA derivative of E. coli K-12 W3110) was used for expression, fermentation and incorporation experiments. In brief, transformed host cells were grown in New Minimal Medium (NMM) which contains 22 mM KH2PO4, 50 mM K2HPO4,
Translational activity of methyl- and aminotryptophan analogs
Barstar pwt, widely used as a model protein for folding studies, contains three Trp residues: Trp38 and Trp44 which are fully or partially solvent exposed and Trp53 which is completely buried inside the hydrophobic core of the protein [30]. Recently, we have discovered that all these three Trp residues of pwt-barstar can be substituted by (4-NH2)Trp and (5-NH2)Trp but not with (6-NH2)Trp and (7-NH2)Trp using the SPI method with the Trp-auxotrophic E. coli ATCC49980 strain as expression host [14]
Barstar folding and the functional role of its Trp residues
In principal, the stabilizing element in all globular proteins is the hydrophobic core. This core in the N-state structure, shielded from the solvent, is probably the most tightly packed form of organic matter [23]. Its close packing provides a unique and constrained conformation, whereas loose packing is often associated with flexible hinges and conformational changes. This high degree of side chain packing in a rigid core of proteins must be established from an unfolded conformation. Two
Acknowledgements
We thank Robert Huber and Luis Moroder for providing us with infrastructure and laboratory space. We are grateful to Peter Göttig and M. Kamran Azim for their help in crystallographic and modeling experiments and Mekdes Debela for help in manuscript preparation. Special thanks from R. G. go to his internship students Franziska Seifert and Erik Hinze. This work was supported by Deutsche Forschungsgemeinschaft (SFB533) and BMBF “BioFuture” program.
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Both authors equally contributed to this work.