Probing the Local Secondary Structure of Human Vimentin with Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy Lishan Liu, † John Hess, ‡ Indra D. Sahu, † Paul G. FitzGerald, ‡ Robert M. McCarrick, † and Gary A. Lorigan* ,† † Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States ‡ Dept of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California 95616, United States *S Supporting Information 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 selectively 2 H-labeled and a nitroxide spin label is strategically placed 1, 2, 3, or 4 amino acids away from the 2 H-labeled amino acid (denoted as i ± 1 to i ± 4, i represents the 2 H- labeled amino acid). ESEEM can detect the dipolar coupling between the nitroxide spin label and 2 H 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, a 2 H ESEEM signal can be detected for i ± 3 and i ± 4 samples, but not for i ± 1 or i ± 2 samples. Several 2 H-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. ■ INTRODUCTION The cytoskeleton system is an integrated network responsible for the mechanical integrity, mobility, and plasticity of the cell. 1,2 The intermediate filament (IF) protein family, as one of key components for the cytoskeleton network, consists of more than 60 members. 3 There has been a growing number of mutations in IF proteins which have been associated with severe muscular, neuronal, and skin diseases. 4 A better understanding of IF protein structures can provide mechanistic insights on their assembly and function. All IF proteins are composed of a highly conserved helical central rod domain, which is flanked by an N-terminal head domain and a C-terminal tail domain as illustrated in Figure 1. 2,5 The central domain was originally predicted to be composed of 4 coiled-coil domains separated by short noncoiled-coil regions. 6 However, recent EPR and X-ray crystallography (XRC) data reveal that rod domain 2A and linker 2 form a parallel helical structure that transitions into a canonical coiled-coil structure around residues 302−305. 5,7,8 Results from previous EPR and XRC studies on rod domains 1B and 2B agree very well with the results of XRC experiments. 7−14 The only significant difference lies in the nonphysiological arrangement of three coiled coil domains in the crystal structure (PDB ID: 1GK4). 13,14 Since these protein segments located near the termini of the truncated protein construct for crystallization, it is highly likely that they do not represent the dominant conformation in vivo. Due to their fibrous nature and self-assembling ability, no X- ray structure of a full-length IF protein has been obtained. However, peptide sequences derived from human vimentin Received: October 4, 2016 Revised: November 10, 2016 Published: November 10, 2016 Figure 1. Carton representation of predicted vimentin structure. The central rod domain emphasized. α-helical rod subdomains 1A, 1B, 2A, and 2B are shown as shaded boxes. Hypothesized nonhelical linker regions L1, L12, and L2 are drawn as thin lines. The region of rod subdomain 2B subject to study is expanded, and the sequence of this region is shown in single-letter amino acid abbreviations. Leu residues at positions 309, 340 are highlighted in bold red. Article pubs.acs.org/JPCB © XXXX American Chemical Society A DOI: 10.1021/acs.jpcb.6b10054 J. Phys. Chem. B XXXX, XXX, XXX−XXX