title: Solution conformation of the extracellular domain of the human tumor necrosis factor receptor probed by Raman and UV-resonance Raman spectroscopy: structural effects of an engineered PEG linker.
abstract: The solution structure of the Escherichia coli-expressed extracellular domain, residues 12-172, of the human 55 kDa type I tumor necrosis factor receptor (TNFR) has been probed by Raman (514.5 nm) and ultraviolet-resonance Raman (244 nm) excitations. The Raman spectra have been collected from both the free TNFR domain and an engineered "dumbbell-like" derivative, consisting of two mutant receptor moieties linked by a 20 kDa polyethylene glycol (PEG) tether. The results demonstrate a TNFR secondary structure which is rich in beta-sheet and deficient in alpha-helix, consistent with the reported X-ray crystal structure of baculovirus expressed receptor complexed with factor beta [Banner, D. W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.-J., Broger, C., Loetscher, H., & Lesslauer, W. (1993) Cell 73, 431-445]. Conversely, the solution structure of TNFR differs from the crystal structure in its distribution of disulfide rotamers and in the orientation of its unique indole side chain (tryptophan-107). These differences are attributed, respectively, to N-terminal truncation and factor binding in the TNFR crystal structure. The tryptophan configuration, which is easily monitored in both Raman and UVRR spectra, is proposed as a potential signal of receptor/factor recognition and binding. Application of the Raman probes to the engineered TNFR dumbbell, which is of interest as a potential therapeutic, shows that TNFR moieties of the dumbbell exhibit secondary structures and side chain environments which are indistinguishable from those of the native, wild-type moiety. The results suggest that the PEGylated dumbbell may function as an effective TNFR drug delivery system without the consequence of a deleterious antigenic response.
authors.affiliation: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri, Kansas City 64110-2499, USA.
authors.forename: R
authors.initials: R
authors.lastname: Tuma
authors.forename: M
authors.initials: M
authors.lastname: Russell
authors.forename: M
authors.initials: M
authors.lastname: Rosendahl
authors.forename: G J
authors.initials: GJ
authors.lastname: Thomas
mesh_terms: D000595:Amino Acid Sequence; D016367:Baculoviridae; D003460:Crystallization; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004926:Escherichia coli; D006801:Humans; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry
chemical_list: D004220:Disulfides; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D012996:Solutions; D011092:Polyethylene Glycols; D014443:Tyrosine; D003553:Cystine; D004229:Dithiothreitol