5EHU

sfGFP mutant with unnatural amino acid 4-azidoethoxy-L-phenylalanine incorporated at the 149 site

Summary for 5EHU

• Entry DOI: 10.2210/pdb5ehu/pdb
• Descriptor: Green fluorescent protein (2 entities in total)
• Functional Keywords: unnatural amino acid, gfp, fluorescent protein
• Biological source: Aequorea victoria (Jellyfish)
• Total number of polymer chains: 2
• Total formula weight: 54034.94

Authors

Tookmanian, E.M.,Phillips-Piro, C.M.,Fenlon, E.E.,Brewer, S.B. (deposition date: 2015-10-28, release date: 2015-12-23, Last modification date: 2023-11-15)

Primary citation

Tookmanian, E.M.,Phillips-Piro, C.M.,Fenlon, E.E.,Brewer, S.H.
Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins.
Chemistry, 21:19096-19103, 2015

PubMed Abstract: An unnatural amino acid, 4-(2-azidoethoxy)-L-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54% overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the azide asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites by using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three-atom linker between the azido group and the aromatic ring of AePhe. An X-ray crystal structure of sfGFP-149-AePhe was solved at 1.45 Å resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP-AePhe constructs efficiently undergo a bioorthogonal strain-promoted click cycloaddition with a dibenzocyclooctyne derivative.

PubMed: 26608683
DOI: 10.1002/chem.201503908

Experimental method

X-RAY DIFFRACTION (1.45 Å)