Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4‑Azidomethyl‑l‑Phenylalanine

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 pN3CH2Ph...

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Published inThe journal of physical chemistry. B Vol. 117; no. 30; pp. 8987 - 8993
Main Authors Bazewicz, Christopher G, Liskov, Melanie T, Hines, Kevin J, Brewer, Scott H
Format Journal Article
LanguageEnglish
Published Washington, DC American Chemical Society 01.08.2013
Subjects
Amino acids
Amino Acyl-tRNA Synthetases - metabolism
Asymmetry
Azides - chemistry
Biological and medical sciences
Coefficients
Extinction
Fluorescence
Fundamental and applied biological sciences. Psychology
Green Fluorescent Proteins - chemistry
Green Fluorescent Proteins - metabolism
Hydrophobic and Hydrophilic Interactions
Molecular biophysics
Nitriles - chemistry
Phenylalanine - analogs & derivatives
Phenylalanine - chemistry
Physical chemistry
Proteins
Spectroscopy, Fourier Transform Infrared
Ultraviolet Rays
Vibration
Local structure
Alanine
Stability
Aminoacid
Illumination
Vibrational mode
Organic compounds
Extinction index
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Summary: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.
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ISSN:1520-6106
1520-5207
DOI:10.1021/jp4052598