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    Keywords: PEPTIDE ; CELLS ; IN-VITRO ; CELL ; human ; VITRO ; PROTEIN ; DOMAIN ; BIOLOGY ; SPECTROSCOPY ; FORM ; SUBUNIT ; MUTATION ; CRYSTAL-STRUCTURE ; STABILITY ; INTERMEDIATE-FILAMENTS ; vimentin ; DIMER ; lamin ; DOMAINS ; ATOMIC-STRUCTURE ; intermediate filament ; SINGLE ; molecular biology ; assembly ; REARRANGEMENT ; coiled coil ; COILED-COIL ; nuclear lamins ; TEMPERATURE ; AMINO-ACID SUBSTITUTIONS ; circular dichroism ; POSITION ; STATE ; biophysical analysis ; CONSENSUS MOTIF ; GCN4 LEUCINE-ZIPPER ; HYDROPHOBIC CORE ; Oligomerisation ; OLIGOMERIZATION STATE ; PROTEIN STRUCTURES
    Abstract: Interestingly, our previously published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric alpha-helical coil instead of the expected dimeric coiled coil. However, the 39-amino-acid-long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim of investigating the molecular criteria that are needed to stabilize a dimeric coiled-coil structure. We have analysed the biophysical properties of the mutants by circular dichroism spectroscopy, analytical ultracentrifugation and X-ray crystallography. All four mutants exhibited an increased stability over the wild type as indicated by a rise in the melting temperature (T-m). At a concentration of 0.1 mg/ml, the T-m of the peptide with the single point mutation Y117L increased dramatically by 46 degrees C compared with the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or a d position did induce the formation of a stable dimer as demonstrated by sedimentation equilibrium experiments. The dimeric oligomerisation state of the Y117L peptide was furthermore confirmed by Xray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when this mutation was introduced into full-length vimentin, filament assembly was completely arrested at the unit-length filament (ULF) level, both in vitro and in cDNA-transfected cultured cells. Therefore, the low propensity of the wild-type coil 1A to form a stable two-stranded coiled coil is most likely a prerequisite for the end-to-end annealing of ULFs into filaments. Accordingly, the coil 1A domains might "switch" from a dimeric alpha-helical coiled coil into a more open structure, thus mediating, within the ULFs, the conformational rearrangements of the tetrameric subunits that are needed for the intermediate filament elongation reaction. (C) 2009 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 19422834
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