TY - JOUR
T1 - Monitoring intermediate filament assembly by small-angle x-ray scattering reveals the molecular architecture of assembly intermediates
AU - Sokolova, Anna V.
AU - Kreplak, Laurent
AU - Wedig, Tatjana
AU - Mücke, Norbert
AU - Svergun, Dmitri I.
AU - Herrmann, Harald
AU - Aebi, Ueli
AU - Strelkov, Sergei V.
PY - 2006/10/31
Y1 - 2006/10/31
N2 - Intermediate filaments (IFs), along with microtubules, microfilaments, and associated cross-bridging proteins, constitute the cytoskeleton of metazoan cells. While crystallographic data on the dimer representing the elementary IF "building block" have recently become available, little structural detail is known about both the mature IF architecture and its assembly pathway. Here, we have applied solution small-angle x-ray scattering to investigate the in vitro assembly of a 53-kDa human IF protein vimentin at pH 8.4 by systematically varying the ionic strength conditions, and complemented these experiments by electron microscopy and analytical ultracentrifugation. While a vimentin solution in 5 mM Tris-HCl (pH 8.4) contains predominantly tetramers, addition of 20 mM NaCl induces further lateral assembly evidenced by the shift of the sedimentation coeficient and yields a distinct octameric intermediate. Four octamers eventually associate into unit-length filaments (ULFs) that anneal longitudinally. Based on the small-angle x-ray scattering experiments supplemented by crystallographic data and additional structural constraints, 3D molecular models of the vimentin tetramer, octamer, and ULF were constructed. Within each of the three oligomers, the adjacent dimers are aligned exclusively in an approximately half-staggered antiparallel A11 mode with a distance of 3.2-3.4 nm between their axes. The ULF appears to be a dynamic and a relatively loosely packed structure with a roughly even mass distribution over its cross-section.
AB - Intermediate filaments (IFs), along with microtubules, microfilaments, and associated cross-bridging proteins, constitute the cytoskeleton of metazoan cells. While crystallographic data on the dimer representing the elementary IF "building block" have recently become available, little structural detail is known about both the mature IF architecture and its assembly pathway. Here, we have applied solution small-angle x-ray scattering to investigate the in vitro assembly of a 53-kDa human IF protein vimentin at pH 8.4 by systematically varying the ionic strength conditions, and complemented these experiments by electron microscopy and analytical ultracentrifugation. While a vimentin solution in 5 mM Tris-HCl (pH 8.4) contains predominantly tetramers, addition of 20 mM NaCl induces further lateral assembly evidenced by the shift of the sedimentation coeficient and yields a distinct octameric intermediate. Four octamers eventually associate into unit-length filaments (ULFs) that anneal longitudinally. Based on the small-angle x-ray scattering experiments supplemented by crystallographic data and additional structural constraints, 3D molecular models of the vimentin tetramer, octamer, and ULF were constructed. Within each of the three oligomers, the adjacent dimers are aligned exclusively in an approximately half-staggered antiparallel A11 mode with a distance of 3.2-3.4 nm between their axes. The ULF appears to be a dynamic and a relatively loosely packed structure with a roughly even mass distribution over its cross-section.
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U2 - 10.1073/pnas.0603629103
DO - 10.1073/pnas.0603629103
M3 - Article
C2 - 17050693
AN - SCOPUS:33750843407
SN - 0027-8424
VL - 103
SP - 16206
EP - 16211
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 44
ER -