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Modulating the bioluminescence emission of photoproteins by in vivo site-directed incorporation of non-natural amino acids.

ABSTRACT: The in vivo incorporation of non-natural amino acids into specific sites within proteins has become an extremely powerful tool for bio- and protein chemists in recent years. One avenue that has yet to be explored, however, is whether or not the incorporation of non-natural amino acids can tune the color of light emitted by bioluminescent proteins, whose light emission mechanisms are more complex and less well understood than their fluorescent counterparts. Bioluminescent proteins are becoming increasingly important in a variety of research fields, such as in situ imaging and the study of protein-protein interactions in vivo, and an increased spectral variety of bioluminescent reporters is needed for further progress. Thus, herein we report the first successful spectral shifting (44 nm) of a bioluminescent protein, aequorin, via the site-specific incorporation of several non-natural amino acids into an integral amino acid position within the aequorin structure in vivo.

SUBMITTER: Rowe L

PROVIDER: S-EPMC2874640 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Modulating the bioluminescence emission of photoproteins by in vivo site-directed incorporation of non-natural amino acids.

Rowe Laura L Ensor Mark M Mehl Ryan R Daunert Sylvia S

ACS chemical biology 20100501 5

The in vivo incorporation of non-natural amino acids into specific sites within proteins has become an extremely powerful tool for bio- and protein chemists in recent years. One avenue that has yet to be explored, however, is whether or not the incorporation of non-natural amino acids can tune the color of light emitted by bioluminescent proteins, whose light emission mechanisms are more complex and less well understood than their fluorescent counterparts. Bioluminescent proteins are becoming in ...[more]

PMID: 20230005

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Project description:Post-translational modifications (PTMs) of protein tyrosine (Tyr) residues can serve as a molecular fingerprint of exposure to distinct oxidative pathways and are observed in abnormally high abundance in the majority of human inflammatory pathologies. Reactive oxidants generated during inflammation include hypohalous acids and nitric oxide-derived oxidants, which oxidatively modify protein Tyr residues via halogenation and nitration, respectively, forming 3-chloroTyr, 3-bromoTyr, and 3-nitroTyr. Traditional methods for generating oxidized or halogenated proteins involve nonspecific chemical reactions that result in complex protein mixtures, making it difficult to ascribe observed functional changes to a site-specific PTM or to generate antibodies sensitive to site-specific oxidative PTMs. To overcome these challenges, we generated a system to efficiently and site-specifically incorporate chloroTyr, bromoTyr, and iodoTyr, and to a lesser extent nitroTyr, into proteins in both bacterial and eukaryotic expression systems, relying on a novel amber stop codon-suppressing mutant synthetase (haloTyrRS)/tRNA pair derived from the Methanosarcina barkeri pyrrolysine synthetase system. We used this system to study the effects of oxidation on HDL-associated protein paraoxonase 1 (PON1), an enzyme with important antiatherosclerosis and antioxidant functions. PON1 forms a ternary complex with HDL and myeloperoxidase (MPO) in vivo. MPO oxidizes PON1 at tyrosine 71 (Tyr71), resulting in a loss of PON1 enzymatic function, but the extent to which chlorination or nitration of Tyr71 contributes to this loss of activity is unclear. To better understand this biological process and to demonstrate the utility of our GCE system, we generated PON1 site-specifically modified at Tyr71 with chloroTyr and nitroTyr in Escherichia coli and mammalian cells. We demonstrate that either chlorination or nitration of Tyr71 significantly reduces PON1 enzymatic activity. This tool for site-specific incorporation of halotyrosine will be critical to understanding how exposure of proteins to hypohalous acids at sites of inflammation alters protein function and cellular physiology. In addition, it will serve as a powerful tool for generating antibodies that can recognize site-specific oxidative PTMs.

Project description:Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents, should make K1 labeling an increasingly viable alternative to cysteine-based methods for SDSL in proteins.

Dataset Information

Modulating the bioluminescence emission of photoproteins by in vivo site-directed incorporation of non-natural amino acids.

Publications

Modulating the bioluminescence emission of photoproteins by in vivo site-directed incorporation of non-natural amino acids.

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