Dataset Information

Sensitive, site-specific, and stable vibrational probe of local protein environments: 4-azidomethyl-L-phenylalanine.

ABSTRACT: We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-L-phenylalanine (pN?CH?Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN?CH?Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN?Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN?CH?Phe to different protein environments to be measured. The photostability of pN?CH?Phe was also measured relative to the photoreactive UAA, pN?Phe.

SUBMITTER: Bazewicz CG

PROVIDER: S-EPMC3769706 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Sensitive, site-specific, and stable vibrational probe of local protein environments: 4-azidomethyl-L-phenylalanine.

Bazewicz Christopher G CG Liskov Melanie T MT Hines Kevin J KJ Brewer Scott H SH

The journal of physical chemistry. B 20130718 30

We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-L-phenylalanine (pN₃CH₂Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN₃CH₂Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN₃Phe). This UAA was genetically incorporated in a site-specific manner utilizing an enginee ...[more]

PMID: 23865850

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Project description:The abundant iron sulfide mineral pyrite has been shown to catalytically produce hydrogen peroxide (H2O2) and hydroxyl radical (.OH) in slurries of oxygenated water. Understanding the formation and fate of these reactive oxygen species is important to biological and ecological systems as exposure can lead to deleterious health effects, but also environmental engineering during the optimization of remediation approaches for possible treatment of contaminated waste streams. This study presents the use of the amino acid phenylalanine (Phe) to monitor the kinetics of pyrite-induced .OH formation through rates of hydroxylation forming three isomers of tyrosine (Tyr) - ortho-, meta-, and para-Tyr. Results indicate that about 50% of the Phe loss results in Tyr formation, and that these products further react with .OH at rates comparable to Phe. The overall loss of Phe appeared to be pseudo first-order in [Phe] as a function of time, but for the first time it is shown that initial rates were much less than first-order as a function of initial substrate concentration, [Phe]o. These results can be rationalized by considering that the effective concentration of .OH in solution is lower at a higher level of reactant and that an increasing fraction of .OH is consumed by Phe-degradation products as a function of time. A simplified first-order model was created to describe Phe loss in pyrite slurries which incorporates the [Phe]o, a first-order dependence on pyrite surface area, the assumption that all Phe degradation products compete equally for the limited supply of highly reactive .OH, and a flux that is related to the release of H2O2 from the pyrite surface (a result of the incomplete reduction of oxygen at the pyrite surface). An empirically derived rate constant, Kpyr, was introduced to describe a variable .OH-reactivity for different batches of pyrite. Both the simplified first-order kinetic model, and a more detailed numerical simulation, yielded results that compare well to the observed kinetic data describing the effects of variations in concentrations of both initial Phe and pyrite. This work supports the use of Phe as a useful probe to assess the formation of .OH in the presence of pyrite, and its possible utility for similar applications with other minerals.

Dataset Information

Sensitive, site-specific, and stable vibrational probe of local protein environments: 4-azidomethyl-L-phenylalanine.

Publications

Sensitive, site-specific, and stable vibrational probe of local protein environments: 4-azidomethyl-L-phenylalanine.

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