title: Probing the Local Secondary Structure of Human Vimentin with Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.
abstract: Previously, an electron spin echo envelope modulation (ESEEM) spectroscopic approach was established to probe the local secondary structure of membrane proteins and peptides utilizing site-directed spin-labeling (SDSL). In this method, the side chain of one amino acid residue is selectively2H-labeled and a nitroxide spin label is strategically placed 1, 2, 3, or 4 amino acids away from the2H-labeled amino acid (denoted as i ± 1 to i ± 4, i represents the2H-labeled amino acid). ESEEM can detect the dipolar coupling between the nitroxide spin label and2H atoms on the amino acid side chain. Due to the periodicity of different secondary structures, different ESEEM patterns can be revealed to probe the structure. For an α-helical structural component, a2H ESEEM signal can be detected for i ± 3 and i ± 4 samples, but not for i ± 1 or i ± 2 samples. Several2H-labeled hydrophobic amino acids have been demonstrated in model system that can be utilized to identify local secondary structures via this ESEEM approach in an extremely efficient fashion. In this study, the ESEEM approach was used to investigate the rod 2B region of the full-length intermediate filament protein human vimentin. Consistent with previous EPR and X-ray crystallography results, our ESEEM results indicated helical structural components within this region. Thus, this ESEEM approach is able to identify α-helical structural components despite the coiled-coil nature of the vimentin structure. The data show that the human vimentin rod 2B adapted a typical α-helical structure around residue Leu309. This result is consistent with the X-ray data from fragmented protein segments and continuous wave EPR data on the full-length vimentin. Finally, the ESEEM data suggested that a local secondary structure slightly different from a typical α-helix was adopted around residue 340.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Lishan
authors.initials: L
authors.lastname: Liu
authors.affiliation: Dept of Cell Biology and Human Anatomy, School of Medicine, University of California , Davis, California 95616, United States.
authors.forename: John
authors.initials: J
authors.lastname: Hess
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Indra D
authors.initials: ID
authors.lastname: Sahu
authors.affiliation: Dept of Cell Biology and Human Anatomy, School of Medicine, University of California , Davis, California 95616, United States.
authors.forename: Paul G
authors.initials: PG
authors.lastname: FitzGerald
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Robert M
authors.initials: RM
authors.lastname: McCarrick
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Gary A
authors.initials: GA
authors.lastname: Lorigan
mesh_terms: D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D014746:Vimentin / Q000737:chemistry*
chemical_list: D014746:Vimentin