title: Probing the effectiveness of spectroscopic reporter unnatural amino acids: a structural study.
abstract: The X-ray crystal structures of superfolder green fluorescent protein (sfGFP) containing the spectroscopic reporter unnatural amino acids (UAAs) 4-cyano-L-phenylalanine (pCNF) or 4-ethynyl-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.
authors.affiliation: Department of Chemistry, Franklin and Marshall College, PO Box 3003, Lancaster, PA 17604-3003, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Dippel
authors.affiliation: Department of Chemistry, Franklin and Marshall College, PO Box 3003, Lancaster, PA 17604-3003, USA.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Olenginski
authors.affiliation: Department of Chemistry, Franklin and Marshall College, PO Box 3003, Lancaster, PA 17604-3003, USA.
authors.forename: Nicole
authors.initials: N
authors.lastname: Maurici
authors.affiliation: Department of Chemistry, Franklin and Marshall College, PO Box 3003, Lancaster, PA 17604-3003, USA.
authors.forename: Melanie T
authors.initials: MT
authors.lastname: Liskov
authors.affiliation: Department of Chemistry, Franklin and Marshall College, PO Box 3003, Lancaster, PA 17604-3003, USA.
authors.forename: Scott H
authors.initials: SH
authors.lastname: Brewer
authors.affiliation: Department of Chemistry, Franklin and Marshall College, PO Box 3003, Lancaster, PA 17604-3003, USA.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Phillips-Piro
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D037402:Hydrozoa / Q000737:chemistry*; D008958:Models, Molecular; D010649:Phenylalanine / Q000031:analogs & derivatives*
chemical_list: D005456:Fluorescent Dyes; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D010649:Phenylalanine
keywords: 4-cyano-l-phenylalanine; 4-ethynyl-l-phenylalanine; green fluorescent protein; unnatural amino acids