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CHARACTERIZATION OF THE LINKER 1–2 REGION IN HUMAN VIMENTIN USING SITE DIRECTED SPIN LABELING AND ELECTRON PARAMAGNETIC RESONANCE
Author(s) -
Hess John F,
Budamagunta Madhu S,
Voss John C,
FitzGerald Paul G
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a96-a
Subject(s) - linker , electron paramagnetic resonance , site directed spin labeling , coiled coil , crystallography , chemistry , dimer , heptad repeat , pulsed epr , nuclear magnetic resonance , peptide sequence , spin echo , magnetic resonance imaging , physics , biochemistry , medicine , organic chemistry , radiology , computer science , operating system , gene
Site Directed Spin Labeling and Electron Paramagnetic Resonance were used to probe residues 281–304 of human vimentin, a region predicted to be a non‐alpha helical linker and the beginning of coiled‐coil domain 2B. EPR analysis of spin‐labeled mutants indicates that a) several residues reside in close proximity, suggesting that adjacent linker regions in a dimer run in parallel, and b) the polypeptide backbone is relatively rigid and inflexible in this region. This region does not show the characteristics of a coiled‐coil as has been identified elsewhere in the molecule. Within this region, spectra from positions 283 and 291 are unique from all others thus far examined. These positions, predicted to be in a non coiled‐coil structure, display a significantly stronger interaction than the a‐d contact positions of coiled‐coil regions. Analysis of the early stages of assembly shows the close apposition and structural rigidity at residues 283 and 291 occurs very early in assembly, and with a relatively sudden onset. These features are inconsistent with hypotheses that envision the linkers as flexible regions, or as looping away from one another, and raise the possibility that the linker may be site at which dimer alignment and/or formation is initiated. Spin labels placed further downstream yield spectra suggesting that the first regular heptad begins at position 302. In conjunction with our previous characterization of region 305–336, and the solved structure of rod 2B from 328–405, the full extent of coiled‐coil domain in rod 2B is now known, spanning from vimentin positions 302–405.