Structural characterization of human vimentin rod 1 and the sequencing of assembly steps in intermediate filament formation in vitro using site-directed spin labeling and electron paramagnetic resonance.
We have previously established the utility of site-directed spin labeling and electron paramagnetic resonance to determine structural relationships among proteins in intact intermediate filaments. Using this same approach we have introduced spin labels at 21 residues between amino acids 169 and 193 in rod domain 1 of human vimentin. The electron paramagnetic resonance spectra provide direct evidence for the coiled coil nature of the vimentin dimer in this region. This finding is consistent with predictions but has never been demonstrated previously. In a previous study we identified residue 348 in the rod domain 2 as one point of overlap between adjacent dimers in intact filaments. In the present study we defined residue 191 in the rod domain 1 as a second point of overlap and established that the dimers are arranged in an anti-parallel and staggered orientation at this site. Finally, by isolating spin-labeled samples at successive stages during the dialysis that lead to filament assembly in vitro, we have been able to establish a sequence of interactions that occurs during in vitro assembly, starting with the alpha helix and loose coiled coil dimer formation, then the formation of tetrameric species centered on residue 191, followed by interactions centered on residue 348 suggestive of octamer or higher order multimer formation. A continuation of this strategy revealed that both 191-191 and 348-348 interactions are present in low ionic strength Tris buffers when vimentin is maintained at the "protofilament" stage of assembly.
Hess
John F
JF
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis 95616, USA.
Budamagunta
Madhu S
MS
Voss
John C
JC
FitzGerald
Paul G
PG
D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry*; D007477:Ions; D009154:Mutation; D017434:Protein Structure, Tertiary; D013113:Spin Labels; D014746:Vimentin / Q000737:chemistry* / Q000235:genetics
D007477:Ions; D013113:Spin Labels; D014746:Vimentin