Unnatural Amino Acid Side Chains Research Articles

Post-translational mutagenesis for installation of natural and unnatural amino acid side chains into recombinant proteins.

Methods for installing natural and unnatural amino acids and their modifications into proteins in a benign and precise manner are highly sought-after in protein science. Here we describe a protocol for 'post-translational mutagenesis' that enables the programmed installation of protein side chains through the use of rapid, mild and operationally simple free-radical chemistry performed on recombinantly expressed and purified proteins. By introduction of protein dehydroalanine (Dha) residues (in this instance, from a unique cysteine residue introduced by site-directed mutagenesis) as free-radical trapping 'tags' for downstream modification, exquisite control over the site of subsequent modification is achieved. Using readily available alkyl halide precursors and simple borohydride salts, alkyl radicals can be generated in aqueous solution. These alkyl radicals react rapidly with protein-bound Dha residues to yield functionalized protein products with new carbon-carbon bonds. Once the Dha is installed, the introduction of the desired functionality is limited only by the requirement for polarity matching of the generated radical with the Dha 'acceptor', the solubility of the alkyl halide precursors in aqueous solution and the kinetics of the reaction itself. For example, methylated derivatives of lysine, arginine and glutamine are readily accessible. Furthermore, as the side chains are constructed chemically, many unnatural modifications can also be directly introduced as part of the side chain, including isotope reporters (19F, 13C) that can be used in biophysical experiments such as protein NMR. From a suitable cysteine mutant of the target protein, the entire procedure for this chemical post-translational mutation takes 2 d and is readily performed by nonchemists.

Enzyme directed formation of un-natural side-chains for covalent surface attachment of proteins

The covalent immobilization of proteins onto surfaces is an essential aspect of several fields of research, including proteomics, sensing, heterogeneous biocatalysis, and more broadly biotechnology. Site-specific, covalent attachment of proteins has been achieved in recent years by the use of expanded genetic codes to produce proteins with controlled placement of un-natural amino acids bearing bio-orthogonal functional groups. Unfortunately, the complexity of developing such systems is impractical for most laboratories; hence, a less complicated approach to generating un-natural amino acid side-chains has been employed. Utilizing a straightforward reaction with formylglycine generating enzyme, we use the site-specific modification of engineered proteins to yield un-natural amino acid side-chains for protein immobilization. Using this approach, we demonstrate the controlled immobilization of various enzymes onto a variety of amine coated surfaces. Our results reveal reusability of the immobilized enzymes via this strategy, and furthermore, we find the activity of the immobilized enzymes to remain even after a month of use indicating significant stability of the linkage.

Incorporation of Unnatural Amino Acids into Trimeric Ion Channels

The incorporation of unnatural amino acid side chains into ion channels has greatly aided the understanding of these physiologically and pharmacologically important membrane proteins. Although the so-called in vivo nonsense suppression method for the incorporation of unnatural amino acids is extremely versatile, not all ion channels appear to be amenable to this experimental approach. In fact, the technique has thus far been primarily applied to pentameric ligand-gated ion channels, and those in the voltage-gated channel family. Here, we show that unnatural derivatives of Phe and Trp can be successfully incorporated into the trimeric ASIC1 cation channel, from the ENaC/degenerin family of ion channels, as well as the P2X2 receptor, an ATP-gated ion channel. Importantly, unnatural derivatives were well tolerated at different positions in both extracellular and transmembrane domains, with rescued channels displaying robust expression and WT-like gating characteristics. The results suggest that these important ion channel families are amenable to novel chemical biology tools such as the incorporation of unnatural amino acids and thus open up new avenues for their study.

Glycated AAV Vectors: Chemical Redirection of Viral Tissue Tropism

A chemical approach for selective masking of arginine residues on viral capsids featuring an exogenous glycation reaction has been developed. Reaction of adeno-associated viral (AAV) capsids with the α-dicarbonyl compound, methylglyoxal, resulted in formation of arginine adducts. Specifically, surface-exposed guanidinium side chains were modified into charge neutral hydroimidazolones, thereby disrupting a continuous cluster of basic amino acid residues implicated in heparan sulfate binding. Consequent loss in heparin binding ability and decrease in infectivity were observed. Strikingly, glycated AAV retained the ability to infect neurons in the mouse brain and were redirected from liver to skeletal and cardiac muscle following systemic administration in mice. Further, glycated AAV displayed altered antigenicity demonstrating the potential for evading antibody neutralization. Generation of unnatural amino acid side chains through capsid glycation might serve as an orthogonal strategy to engineer AAV vectors displaying novel tissue tropisms for gene therapy applications.

Corrections to Unnatural Amino Acid Side Chains as S1, S1′, and S2′ Probes Yield Cationic Antimicrobial Peptides with Stability toward Chymotryptic Degradation

Corrections to Unnatural Amino Acid Side Chains as S1, S1′, and S2′ Probes Yield Cationic Antimicrobial Peptides with Stability toward Chymotryptic Degradation

Unnatural Amino Acid Side Chains as S1, S1′, and S2′ Probes Yield Cationic Antimicrobial Peptides with Stability toward Chymotryptic Degradation

This work describes how the systematic incorporation of a range of unnatural amino acid derivatives in the P1, P1', and P2' positions allows for the generation of short lactoferricin based cationic antimicrobial peptides with a stability toward chymotryptic degradation. The necessary pharmacophore sets the peptides up for degradation by chymotrypsin, and a heavily truncated native tripeptide was rapidly digested despite its short sequence. Degradation studies indicated that increased half-lives could be obtained by altering the binding to each subsite surrounding the active site without sacrificing the antimicrobial activity. Important structural and mechanistic features were revealed in a fashion not feasible through the use of native peptide substrates. The results, which are generally applicable to a range of relevant peptides, further show that not only the S1 pocket, but also to the notably less studied S1' site can be used to control the proteolytic stability by incorporating different analogues of tryptophan and arginine.

The controlled introduction of multiple negative charge at single amino acid sites in subtilisin Bacillus lentus

The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). A series of mono-, di- and triacidic acid methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1′ site. Kinetic parameters for these chemically modified mutant (CMM) enzymes were determined at pH 8.6 under conditions which ensured complete ionization of the unnatural amino acid side-chains introduced. The presence of up to three negative charges in the S1, S1′ and S2 subsites of SBL resulted in up to 11-fold lowered activity, possibly due to interference with oxyanion stabilization of the transition state of the hydrolytic reactions catalyzed. Each unit increase in negative charge resulted in a raising of KM and a reduction of kcat. However, no upper limit was observed for increases in KM, whereas decreases in kcat reached a limiting value. Comparison with sterically similar but uncharged CMMs revealed that electrostatic effects of negative charges at positions 62, 156 and 217 are detrimental, but are beneficial at position 166. These results indicate that the ground-state binding of SBL to the standard substrate, Suc-AAPF-pNA, to SBL is reduced, but without drastic attenuation of catalytic efficiency, and show that SBL tolerates high levels of charge at single sites.

Nucleoside monophosphate kinases: structure, mechanism, and substrate specificity.

The catalytic mechanisms of adenylate kinase, guanylate kinase, uridylate kinase, and cytidylate kinase are reviewed in terms of kinetic and structural information that has been obtained in recent years. All four kinases share a highly related tertiary structure, characterized by a central five-stranded parallel beta-sheet with helices on both sides, as well as the three regions designated as the CORE, NMPbind, and LID domains. The catalytic mechanism continues to be refined to higher levels of resolution by iterative structure-function studies, and the strengths and limitations of site-directed mutagenesis are well illustrated in the case of adenylate kinase. The identity and roles of active site residues now appear to be resolved, and this review describes how specific site substitutions with unnatural amino acid side-chains have proven to be a major advance. Likewise, there is mounting evidence that phosphoryl transfer occurs by an associative transition state, based on (a) the stereochemical course of phosphoryl transfer, (b) geometric considerations, (c) examination of likely electronic distributions, (d) the orientation of the phosphoryl acceptor relative to the phosphoryl being transferred, (e) the most likely role of magnesium ion, (f) the lack of restricted access of solvent water, and (g) the results of oxygen-18 kinetic isotope. effect experiments.

Chemical Modifications at a Single Site Can Induce Significant Shifts in the pH Profiles of a Serine Protease†

Developing the ability to vary the pH−activity profile of an enzyme in a controlled manner has been a long sought-after goal. Such tailoring provides important insights into mechanism and permits optimization of enzyme performance in organic synthesis applications. The most successful approaches to date toward altering pH−activity profiles of enzymes have employed either site-directed mutagenesis or chemical modification to alter enzyme surface charge. We now report that, by combining these two methodologies, dramatic pKa changes can be induced in the serine protease subtilisin B. lentus. In particular, site specific incorporation of unnatural amino acid side chains by the following strategy, WT → Asn62Cysmutant + H3C−SO2−S−R → Asn62Cys-S−R, where R may be infinitely variable, has demonstrated that pKa shifts of up to 0.72 unit are achievable and are accompanied by significant activity enhancements. The most dramatic pKa shifts are caused by chemical modification with hydrophobic aliphatic moieties. A lin...

Use of Site-directed Chemical Modification to Study an Essential Lysine in Escherichia coli Leader Peptidase

Escherichia coli leader peptidase, which catalyzes the cleavage of signal peptides from pre-proteins, is an essential, integral membrane serine peptidase that has its active site residing in the periplasmic space. It contains a conserved lysine residue that has been proposed to act as the general base, abstracting the proton from the side chain hydroxyl group of the nucleophilic serine 90. To help elucidate the role of the essential lysine 145 in the activity of E. coli leader peptidase, we have combined site-directed mutagenesis and chemical modification methods to introduce unnatural amino acid side chains at the 145-position. We show that partial activity can be restored to an inactive K145C leader peptidase mutant by reacting it with 2-bromoethylamine.HBr to produce a lysine analog (gamma-thia-lysine) at the 145-position. Modification with the reagents 3-bromopropylamine.HBr and 2-mercaptoethylamine also allowed for partial restoration of activity showing that there is some flexibility in the length requirements of this essential residue. Modification with (2-bromoethyl)trimethylammonium.Br to form a positively charged, nontitratable side chain at the 145-position failed to restore activity to the inactive K145C leader peptidase mutant. This result, along with an inactive K145R mutant result, supports the claim that the lysine side chain at the 145-position is essential due to its ability to form a hydrogen bond(s) or to act as a general base rather than because of an ability to form a critical salt bridge. We find that leader peptidase processes the pre-protein substrate, pro-OmpA nuclease A, with maximum efficiency at pH 9.0, and apparent pKa values for titratable groups at approximately 8.7 and 9.3 are revealed. We show that the lysine modifier maleic anhydride inhibits leader peptidase by reacting with lysine 145. The results of this study are consistent with the hypothesis that the lysine at the 145-position of leader peptidase functions as the active site general base. A model of the active site region of leader peptidase is presented based on the structure of the E. coli UmuD', and a mechanism for bacterial leader peptidase is proposed.

Mobile unnatural amino acid side chains in the core of staphylococcal nuclease.

The structures of several variants of staphylococcal nuclease with long flexible unnatural amino acid side chains in the hydrophobic core have been determined by X-ray crystallography. The unnatural amino acids are disulfide moieties between the lone cysteine residue in V23C nuclease and methane, ethane, 1-n-propane, 1-n-butane, 1-n-pentane, and 2-hydroxyethyl thiols. We have examined changes in the core packing of these mutants. Side chains as large as the 1-n-propyl cysteine disulfide can be incorporated without perturbation of the structure. This is due, in part, to cavities present in the wild-type protein. The longest side chains are not well defined, even though they remain buried within the protein interior. These results suggest that the enthalpy-entropy balance that governs the rigidity of protein interiors favors tight packing only weakly. Additionally, the tight packing observed normally in protein interiors may reflect, in part, the limited numbers of rotamers available to the natural amino acids.