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J Mol Biol. 2004 Jun 25;340(1):97-114.

Molecular and biophysical characterization of assembly-starter units of human vimentin.

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1
Division of Biophysics of Macromolecules, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.

Abstract

We have developed an assembly protocol for the intermediate filament (IF) protein vimentin based on a phosphate buffer system, which enables the dynamic formation of authentic IFs. The advantage of this physiological buffer is that analysis of the subunit interactions by chemical cross-linking of internal lysine residues becomes feasible. By this system, we have analyzed the potential interactions of the coiled-coil rod domains with one another, which are assumed to make a crucial contribution to IF formation and stability. We show that headless vimentin, which dimerizes under low salt conditions, associates into tetramers of the A(22)-type configuration under assembly conditions, indicating that one of the effects of increasing the ionic strength is to favor coil 2-coil 2 interactions. Furthermore, in order to obtain insight into the molecular interactions that occur during the first phase of assembly of full-length vimentin, we employed a temperature-sensitive variant of human vimentin, which is arrested at the "unit-length filament" (ULF) state at room temperature, but starts to elongate upon raising the temperature to 37 degrees C. Most importantly, we demonstrate by cross-linking analysis that ULF formation predominantly involves A(11)-type dimer-dimer interactions. The presence of A(22) and A(12) cross-linking products in mature IFs, however, indicates that major rearrangements do occur during the longitudinal annealing and radial compaction steps of IF assembly.

PMID:
15184025
DOI:
10.1016/j.jmb.2004.04.039
[Indexed for MEDLINE]
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