Methods for converting cysteine to dehydroalanine on peptides and proteins

Chalker, Justin M.; Gunnoo, Smita B.; Boutureira, Omar; Gerstberger, Stefanie; Fernandez-González, Marta; Bernardes, Gonçalo J. L.; Griffin, Laura; Hailu, Hanna; Schofield, Christopher J.; Davis, Benjamin G.

doi:10.1039/c1sc00185j

Cited by 316 publications

(423 citation statements)

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“…This highlights not only the importance of reducing the total time of heating treatment but also the effect of increasing the electric field applied, as well as the combination of the two effects. An additional non-thermal effect may be explained by unfolding transitions or conformational disturbances of β-lg promoted during its denaturation pathways or by a possible enhancement of oxidative elimination of cysteine to dehydroalanine, also known by a desulfurization (Chalker et al 2011). Despite a trend for reduction of free SH being observed when the other OH treatments were applied (i.e., 6 V cm −1 for CUT of 10 and 100 s and 12 V cm −1 for a CUT of 100 s), these effects were not statistically significant (p > 0.05).…”

Section: Resultsmentioning

confidence: 99%

Production of Whey Protein-Based Aggregates Under Ohmic Heating

Pereira

Rodrigues

Ramos

et al. 2015

Food Bioprocess Technol

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Formation of whey protein isolate protein aggregates under the influence of moderate electric fields upon ohmic heating (OH) has been monitored through evaluation of molecular protein unfolding, loss of its solubility, and aggregation. To shed more light on the microstructure of the protein aggregates produced by OH, samples were assayed by transmission electron microscopy (TEM). Results show that during early steps of an OH thermal treatment, aggregation of whey proteins can be reduced with a concomitant reduction of the heating charge-by reducing the comeup time (CUT) needed to reach a target temperature-and increase of the electric field applied (from 6 to 12 V cm −1 ). Exposure of reactive free thiol groups involved in molecular unfolding of β-lactoglobulin (β-lg) can be reduced from 10 to 20 %, when a CUT of 10 s is combined with an electric field of 12 V cm −1 .Kinetic and multivariate analysis evidenced that the presence of an electric field during heating contributes to a change in the amplitude of aggregation, as well as in the shape of the produced aggregates. TEM discloses the appearance of small fibrillar aggregates upon the influence of OH, which have recognized potential in the functionalization of food protein networks. This study demonstrated that OH technology can be used to tailor denaturation and aggregation behavior of whey proteins due to the presence of a constant electric field together with the ability to provide a very fast heating, thus overcoming heat transfer limitations that naturally occur during conventional thermal treatments.

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Section: Resultsmentioning

confidence: 99%

Production of Whey Protein-Based Aggregates Under Ohmic Heating

Pereira

Rodrigues

Ramos

et al. 2015

Food Bioprocess Technol

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“…2). The chemical modification procedure to introduce the Nca relies on the conversion of a cysteine into dehydroalanine by a bis-alkylation/ elimination sequence (27,36), followed by Michael addition of a thiol to introduce the new Nca side chain. The S. aureus NAL naturally contains no cysteines, making it an ideal target protein for chemical introduction of Ncas (36).…”

Section: Resultsmentioning

confidence: 99%

“…The equivalent positions in the S. aureus NAL based on structural alignment were then mutated into cysteine residues by site-directed mutagenesis, and expressed and purified as previously described (36). The Ncas were then incorporated by first converting the cysteine to dehydroalanine (Dha) using 2,5-dibromohexan-1,6-diamide and then Michael addition with 13 different thiols resulting in installation of 13 different Ncas at each of the 12 positions (27) (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…In addition, in some cases, Nature has exploited noncanonical amino acids (Ncas) in catalysis to extend its catalytic repertoire: for example, the quinones TPQ, LTQ, TTQ, and CTQ, respectively, in amine oxidase, lysyl oxidase, methylamine dehydrogenase, and quinohemoprotein amine dehydrogenase (17); a pyruvoyl group in some histidine, arginine, aspartate, and Sadenosylmethionine decarboxylases (18); formyl glycine residues in type I sulfatases (19); and 4-methylideneimidazole-5-one (MIO) in ammonia lyases and 2,3-aminomutases (20). These Ncas are vital for catalysis and arise through posttranslational modifications of the polypeptide chain (21, 22), allowing access to chemistries not otherwise provided by the 20 proteogenic amino acids.Technologies for the protein engineer to incorporate Ncas into proteins at specifically chosen sites, either by genetic means (23)(24)(25) or by chemical modification (26,27), have recently been developed. These approaches are powerful because, unlike traditional protein engineering with the 20 canonical amino acids, protein engineering with Ncas has almost unlimited novel side-chain structures and chemistries from which to choose.…”

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confidence: 99%

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Extending enzyme molecular recognition with an expanded amino acid alphabet

Windle

Simmons

Ault

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties.protein engineering | aldolases | chemical modification E nzymes are phenomenally powerful catalysts that increase reaction rates by up to 10 18 -fold (1, 2), and a new era of enzyme applications has been opened by the advancement of protein engineering and directed evolution to provide new, or improved, enzymes for industrial biocatalysis. Enzymes are attractive catalysts because they are highly selective, carrying out regio-, chemo-, and stereoselective reactions that are challenging for conventional chemistry. Moreover, enzymes are efficient catalysts, function under mild conditions with relatively nontoxic reagents, and enable the production of relatively pure products, minimizing waste generation. In recent years, there has been much success in engineering enzymes for desired reactions (3-5) using methods such as rational protein engineering (6-8), directed evolution (9-11), and, most recently, computational enzyme design (12-15).Enzymes found in Nature achieve catalysis using active sites generally composed of only 20 canonical amino acids, which are encoded at the genetic level, plus the rarer selenocysteine and 1-pyrrolysine. However, many enzymes also rely on one or more of 27 small organic cofactor molecules and/or 13 metal ions for their function (16). In addition, in some cases, Nature has exploited noncanonical amino acids (Ncas) in catalysis to extend its catalytic repertoire: for example, the quinones TPQ, LTQ, TTQ, and CTQ, respectively, in ...

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“…Dha can be accessed via a number of routes: elimination of active-site serines, the oxidative elimination of unnatural selenocysteine amino acids 163,164 or through the milder oxidative elimination of cysteine with sulfonylhydroxylamine reagents 165 . All can prove efficient but occasional side reactions in all recently prompted the development of a bis-alkylation method: cyclic sulfoniums can be eliminated to form Dha under strikingly mild conditions 166 .…”

Section: Review Nature Communications | Doi: 101038/ncomms5740mentioning

confidence: 99%