Nicolet, Stefan. Coil 2 of intermediate filaments: its complete structure and impact of desminopathy-related mutations. 2011, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_9509
Within this thesis, the dimer structure of vimentin was further investigated using a ‘divide-and-conquer’ approach (Strelkov, 2001). For this, seven different vimentin (D1-D7) fragments covering the predicted linkers L12 and L2 were expressed in E. coli and screened towards crystallization. One of them, the fragment D3, yields crystals suitable for X-ray diffraction. The resulting 2.4Å structure covers the regions of the vimentin molecule formerly designated as coil 2A, linker L2 and the beginning of coil 2B. We show that this region forms a single, contiguous [alpha]-helix and thus, there is no linker L2. Further, the N-terminal part of this fragment starting with Pro263 and ending with Ala302 is not twisted and should be designated as a parallel [alpha]-helical bundle rather than a coiled-coil. The rest of coil 2 is a regular left-handed coiled-coil with the exception of a small unwound region, the stutter, at position 351. The considerable overall sequence similarity of D3 to other IFs (80% for NFL and 60% for keratin types and nuclear lamins) clearly suggests a conserved structural feature to all IF proteins.
We also investigate the impact of some myopathic point mutations in the IF desmin. For this, desmin wild-type (wt) and six mutant fragments (L345P, A360P, L370P, L385P, deltaN366 and E401S) homologous to the previously crystallized vimentin cys2 and lamin A lam1 fragments were expressed in E. coli. With analytical ultracentrifugation and gel-filtration profiles, we show that wt and L345P mutant fragment form dimers in solution, whereas the mutant fragments A360P and E401S are mostly monomeric. Further, we show by circular dichroism that all our fragments were [alpha]-helical. Since our attempts to crystallize these fragments were unsuccessful, we decided to investigate the desmin wt assembly versus desmin myopathic mutants by small angle X-ray scattering (SAXS). For this, we triggered full-length desmin assembly by adding salts to a low ionic strength buffer known to prevent desmin polymerization. Our SAXS measurements in low ionic strength buffer show that almost all mutants are similar to desmin wt, but exhibit more variability with increased ionic strength buffers. In high ionic strength buffer, about half of our samples turned into gel and could not be analyzed by SAXS. Based on SAXS measurements, we were able to classify our mutant into three groups; one group of desmin mutants (L345P, R406W, R350P, A360P, deltaN366, A377P, N342D) behaves as the wt, a second mutant group (E413K, A357P, deltaE359-S361, L385P, E245D, A213V, D399Y, I451M) has an increased assembly upon ionic strength compared to desmin wt and a third group is composed of two outlier mutants (Q389P forming a gel in a low ionic strength buffer and L370P having a broader cross-section diameter than wt already in tetramer buffer).
|Advisors:||Strelkov, Sergei V.|
|Committee Members:||Herrmann, Harald and Aebi, Ueli|
|Faculties and Departments:||05 Faculty of Science > Departement Umweltwissenschaften > Institut für Meteorologie, Klimatologie und Fernerkundung > Meteorologie (Parlow)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||79 S.|
|Last Modified:||30 Jun 2016 10:42|
|Deposited On:||19 Jul 2011 14:45|
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