title: Differentiation of the local structure around tryptophan 51 and 64 in recombinant human erythropoietin by tryptophan phosphorescence.
abstract: Recombinant human erythropoietin is a 4-helix bundle, glycosylated cytokine containing three tryptophan residues at positions 51, 64 and 88 whose phosphorescence emission may represent a sensitive probe of the structure at multiple sites near or at the protein surface. This report characterizes the phosphorescence properties (spectral energy, thermal spectral relaxation and phosphorescence lifetime), from low temperature glasses to ambient temperature, of the native protein plus that of three single point mutation analogs where each Trp was replaced by Phe. The structural information inferred from the phosphorescence parameters was essentially in good agreement with the structure of the Escherichia coli-produced nonglycosylated protein determined by nuclear magnetic resonance (Cheetham et al., Nat Struct Biol [1998] 5:861). The results showed that the fluorescence and phosphorescence spectra of the native protein were entirely due to independent contributions of Trp51 and Trp64 and that Trp88 was quenched under all conditions. The phosphorescence emissions of Trp51 and Trp64 were differentiated by their unique spectra at 77 K with Trp64 exhibiting an unusually blueshifted spectrum likely due to the attractive interaction of Arg110 and Lys116 with the ground state dipole of Trp64. In the native protein the room temperature phosphorescence lifetime of Trp64 was relatively short with a time of 1.62 ms whereas the lifetime of Trp51 was five-fold longer. Characterization of the single point mutation analogs showed that each lifetime was composed of multiple components revealing the presence of multiple stable conformations of the protein at these surface sites.
authors.affiliation: Department of Pharmaceutics, Amgen, Inc., Thousand Oaks, CA, USA. bkerwin@amgen.com
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Kerwin
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Aoki
authors.forename: Margherita
authors.initials: M
authors.lastname: Gonelli
authors.forename: Giovanni B
authors.initials: GB
authors.lastname: Strambini
mesh_terms: D002942:Circular Dichroism; D004921:Erythropoietin / Q000032:analysis / Q000737:chemistry*; D005898:Glass / Q000737:chemistry; D006801:Humans; D049449:Luminescence; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D015335:Molecular Probes / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D015335:Molecular Probes; D011994:Recombinant Proteins; D004921:Erythropoietin; D014364:Tryptophan