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J Phys Chem B. 2013 Aug 1;117(30):8987-93. doi: 10.1021/jp4052598. Epub 2013 Jul 18.

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

Author information

1
Department of Chemistry, Franklin & Marshall College, Lancaster, Pennsylvania 17604-3003, USA.

Abstract

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 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 pN₃CH₂Phe to different protein environments to be measured. The photostability of pN₃CH₂Phe was also measured relative to the photoreactive UAA, pN₃Phe.

PMID:
23865850
PMCID:
PMC3769706
DOI:
10.1021/jp4052598
[Indexed for MEDLINE]
Free PMC Article

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