A Genetically Encoded aza-Michael Acceptor for Covalent Cross-Linking of Protein–Receptor Complexes

Furman, Jennifer L.; Kang, Mingchao; Choi, Sei-hyun; Cao, Yu; Wold, Erik D.; Sun, Sophie; Smider, Vaughn V.; Schultz, Peter G.; Kim, Chan Hyuk

doi:10.1021/ja502851h

Cited by 102 publications

(93 citation statements)

References 45 publications

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“…These longer and more flexible linkages should make it much easier to cross-link sites within proteins. Indeed, it has recently been shown that electrophilic amino acids with extended structures can selectively stabilize proteins and protein complexes by intra-and intermolecular cross-links, respectively (13,(23)(24)(25). However, the use of electrophilic NCAAs to form noncanonical cross-links in a bacterial selection system can be complicated by cellular toxicity, irreversible reactions, reaction rates, and scavenging by intracellular nucleophiles.…”

Section: Discussionmentioning

confidence: 99%

Enhancing protein stability with extended disulfide bonds

Liu

Wang

Luo

et al. 2016

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

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157

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Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9°C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.noncanonical amino acids | extended disulfide bonds | β-lactamase | thermostability | evolutionary advantage

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Section: Discussionmentioning

confidence: 99%

Enhancing protein stability with extended disulfide bonds

Liu

Wang

Luo

et al. 2016

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

Self Cite

157

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“…Proximity-enabled reactivity of ''tuned'' electrophilic NNAAs towards nucleophilic natural amino acids can also be used to probe protein-protein interaction. Selective formation of irreversible covalent bond between a specific lysine of a cancer biomarker ErbB2 and VSF genetically incorporated into anti-ErbB2 Fab was confirmed in vitro and in vivo (Furman et al, 2014). This type of NNAAs has a promise for identifying unknown interacting partners of a target protein, especially those with a large dissociation constant.…”

Section: Biochemical Analysismentioning

confidence: 78%

“…As a result of UV irradiation, a covalently-linked GST dimer was formed when BZF was placed in the dimer interface, but no crosslinking occurred when placed outside the interface. Functional groups targeting specific natural amino acids in a spontaneous and proximity-enhanced manner have been genetically encoded through evolved pairs of amber suppressor tRNA/aaRS (VSF, BCK and ACK; Figure 2) Furman et al, 2014;Xiang et al, 2013;Xiang et al, 2014). These NNAAs have electrophilic residues that selectively react with proximal nucleophiles such as lysine, histidine and cysteine ( Figure 1).…”

Section: Proximity-enabled Bond Formation With a Natural Amino Acidmentioning

confidence: 99%

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

Lim

Kwon

2015

Critical Reviews in Biotechnology

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The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.

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“…Unless otherwise mentioned, all other chemicals were purchased from Sigma-Aldrich and used without further purification. AcrF was synthesized as described (47). Absorbance spectra were measured with a Hewlett-Packard 8453 UV-visible spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

Exploring the potential impact of an expanded genetic code on protein function

Xiao

Nasertorabi

Choi

et al. 2015

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

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With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing k cat , but not significantly affecting K M . To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216-AcrF mutation leads to conformational changes in key active site residues-both in the free enzyme and upon formation of the acylenzyme intermediate-that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.noncanonical amino acid | beta-lactamase | catalytic activity | evolutionary advantage | conformational effects

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A Genetically Encoded aza-Michael Acceptor for Covalent Cross-Linking of Protein–Receptor Complexes

Cited by 102 publications

References 45 publications

Enhancing protein stability with extended disulfide bonds

Enhancing protein stability with extended disulfide bonds

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

Exploring the potential impact of an expanded genetic code on protein function

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