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Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids.
RSC Adv. 2015; 5(2):1274-1281.RA

Abstract

Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-L-phenylalanine (1) and 4-azidomethyl-L-phenylalanine (2), form a series of unnatural amino acids (UAAs) containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine. These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein environments. Three of the four azido-modified phenylalanine residues were successfully genetically incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity. SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP indicated that the azide groups were hydrated in the protein.

Authors+Show Affiliations

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

Pub Type(s)

Journal Article

Language

eng

PubMed ID

26478813

Citation

Tookmanian, Elise M., et al. "Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids." RSC Advances, vol. 5, no. 2, 2015, pp. 1274-1281.
Tookmanian EM, Fenlon EE, Brewer SH. Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids. RSC Adv. 2015;5(2):1274-1281.
Tookmanian, E. M., Fenlon, E. E., & Brewer, S. H. (2015). Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids. RSC Advances, 5(2), 1274-1281.
Tookmanian EM, Fenlon EE, Brewer SH. Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids. RSC Adv. 2015;5(2):1274-1281. PubMed PMID: 26478813.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids. AU - Tookmanian,Elise M, AU - Fenlon,Edward E, AU - Brewer,Scott H, Y1 - 2014/12/02/ PY - 2015/10/20/entrez PY - 2015/10/20/pubmed PY - 2015/10/20/medline KW - Azide Vibrational Reporters KW - Infrared Spectroscopy KW - Unnatural Amino Acids KW - superfolder Green Fluorescent Protein SP - 1274 EP - 1281 JF - RSC advances JO - RSC Adv VL - 5 IS - 2 N2 - Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-L-phenylalanine (1) and 4-azidomethyl-L-phenylalanine (2), form a series of unnatural amino acids (UAAs) containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine. These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein environments. Three of the four azido-modified phenylalanine residues were successfully genetically incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity. SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP indicated that the azide groups were hydrated in the protein. SN - 2046-2069 UR - https://www.unboundmedicine.com/medline/citation/26478813/Synthesis_and_Protein_Incorporation_of_Azido_Modified_Unnatural_Amino_Acids_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/26478813/ DB - PRIME DP - Unbound Medicine ER -
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