Probing the effectiveness of spectroscopic reporter unnatural amino acids: a structural study. Issue 1 (1st January 2016)

• Record Type: Journal Article
• Authors: Dippel, Andrew B.; Olenginski, Gregory M.; Maurici, Nicole; Liskov, Melanie T.; Brewer, Scott H.; Phillips-Piro, Christine M.
• Is Part Of: Acta crystallographica. Volume 72:Issue 1(2016)
• Page Start: 121
• Page End: 130
• Publication Date: 2016-01-01
• Subjects: 4-cyano- 4‐cyano‐l l-phenylalanine ‐phenylalanine -- 4-ethynyl- 4‐ethynyl‐l l-phenylalanine ‐phenylalanine -- unnatural amino acids unnatural amino acids -- green fluorescent protein green fluorescent protein; X-ray crystallography -- Periodicals; Crystallography -- Periodicals; Molecular biology -- Periodicals; Molecular structure -- Periodicals; Biomolecules -- Structure -- Periodicals; Cytology -- Periodicals; Biomolecules -- Structure; Crystallography; Cytology; Molecular biology; Molecular structure; X-ray crystallography; Periodicals; 548
• DOI: 10.1107/S2059798315022858
• Languages: English
• ISSNs: 2059-7983

Abstract

Abstract : The structural implications of incorporating spectroscopic reporter unnatural amino acids into proteins is explored using X‐ray crystallography. Four protein crystal structures with 4‐cyano‐l l ‐phenylalanine or 4‐ethynyl‐l l ‐phenylalanine incorporated into green fluorescent protein at two unique sites are described. Abstract : The X‐ray crystal structures of superfolder green fluorescent protein (sfGFP) containing the spectroscopic reporter unnatural amino acids (UAAs) 4‐cyano‐l l ‐phenylalanine (pCNF) or 4‐ethynyl‐l l ‐phenylalanine (pCCF) at two unique sites in the protein have been determined. These UAAs were genetically incorporated into sfGFP in a solvent‐exposed loop region and/or a partially buried site on the β‐barrel of the protein. The crystal structures containing the UAAs at these two sites permit the structural implications of UAA incorporation for the native protein structure to be assessed with high resolution and permit a direct correlation between the structure and spectroscopic data to be made. The structural implications were quantified by comparing the root‐mean‐square deviation (r.m.s.d.) between the crystal structure of wild‐type sfGFP and the protein constructs containing either pCNF or pCCF in the local environment around the UAAs and in the overall protein structure. The results suggest that the selective placement of these spectroscopic reporter UAAs permits local protein environments to be studied in a relatively nonperturbative fashion with site‐specificity.