Stabilization of vimentin coil2 fragment via an engineered disulfide

Stabilization of vimentin coil2 fragment via an engineered disulfide

Cytoskeletal intermediate filaments (IFs) assemble from the elementary dimers based on a segmented α-helical coiled-coil (CC) structure. Crystallographic studies of IF protein fragments remain the main route to access their atomic structure. To enable crystallization, such fragments must be sufficie...

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Bibliographic Details
Published in:Journal of structural biology Vol. 177; no. 1; pp. 46 - 53
Main Authors: Chernyatina, A.A., Strelkov, S.V.
Format: Journal Article
Language:English
Published: United States Elsevier Inc 2012
Subjects:
Amino Acid Sequence
Coiled coil
Crystallization
Crystallography, X-Ray
Dimerization
Disulfides - analysis
Disulfides - chemistry
Engineered disulfide
Humans
Intermediate filaments
Models, Molecular
Molecular Sequence Data
Protein Multimerization
Protein Structure, Secondary
Protein Structure, Tertiary
Vimentin - analysis
Vimentin - metabolism
Vimentin - ultrastructure
CC
TEV
Intermediate filaments
Crystallization
Engineered disulfide
SeMet
SDS–PAGE
PDB
Tris
Coiled coil
MAD
OD
IPTG
IF
Dimerization
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Summary:Cytoskeletal intermediate filaments (IFs) assemble from the elementary dimers based on a segmented α-helical coiled-coil (CC) structure. Crystallographic studies of IF protein fragments remain the main route to access their atomic structure. To enable crystallization, such fragments must be sufficiently short. As a consequence, they often fail to assemble into the correct CC dimers. In particular, human vimentin fragment D3 corresponding to the first half of coil2 (residues 261–335) stays monomeric in solution. We have induced its dimerization via introducing a disulfide link between two cysteines engineered in the hydrophobic core of the CC close to its N-terminus. The 2.3 Å crystal structure of the D3st (stabilized) fragment reveals a mostly parallel α-helical bundle structure in its N-terminal half which smoothly continues into a left-handed CC towards the C-terminus. This provides a direct evidence for a continuously α-helical structure of the coil2 segment and disproves the previously suggested existence of linker L2 separating it into two left-handed CCs. The general principles of CC dimer stabilization by disulfide introduction are also discussed.
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ISSN:1047-8477
1095-8657
DOI:10.1016/j.jsb.2011.11.014