RESUMO
The selective generation of covalent bonds between and within proteins would provide new avenues for studying protein function and engineering proteins with new properties. New covalent bonds were genetically introduced into proteins by enabling an unnatural amino acid (Uaa) to selectively react with a proximal natural residue. This proximity-enabled bioreactivity was expanded to a series of haloalkane Uaas. Orthogonal tRNA/synthetase pairs were evolved to incorporate these Uaas, which only form a covalent thioether bond with cysteine when positioned in close proximity. By using the Uaa and cysteine, spontaneous covalent bond formation was demonstrated between an affibody and its substrate Z protein, thereby leading to irreversible binding, and within the affibody to increase its thermostability. This strategy of proximity-enabled protein crosslinking (PEPC) may be generally expanded to target different natural amino acids, thus providing diversity and flexibility in covalent bond formation for protein research and protein engineering.
Assuntos
Alcanos/química , Aminoácidos/metabolismo , Halogênios/química , Aminoácidos/química , Aminoacil-tRNA Sintetases/metabolismo , Cisteína/química , Cisteína/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Ligação Proteica , Engenharia de Proteínas , RNA de Transferência/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The ability to reversibly control protein structure and function with light would offer high spatiotemporal resolution for investigating biological processes. To confer photoresponsiveness on general proteins, we genetically incorporated a set of photoswitchable click amino acids (PSCaas), which contain both a reversible photoswitch and an additional click functional group for further modifications. Orthogonal tRNA-synthetases were evolved to genetically encode PSCaas bearing azobenzene with an alkene, keto, or benzyl chloride group in E. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity-enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation.
Assuntos
Aminoácidos/química , Escherichia coli/metabolismo , Química Click , Código Genético , Conformação Molecular , Optogenética , Engenharia de ProteínasRESUMO
Unnatural amino acids (UAAs) containing conjugated ring systems are of interest for their optical properties. Until now, such bulky and planar UAAs could not be incorporated into proteins using the pyrrolysyl tRNA/synthetase shuttling system. Using the "small-intelligent" approach to construct a highly diverse library, we evolved novel synthetases specific for two such UAAs and incorporated them into proteins in E. coli and mammalian cells.
Assuntos
Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Biblioteca Gênica , Aminoácidos/química , Animais , Evolução Molecular Direcionada , Escherichia coli/genética , Código Genético , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Mioglobina/genética , Mioglobina/metabolismo , Especificidade por Substrato , Baleias/genéticaRESUMO
Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability.