Stability profile of vimentin rod domain

Intermediate filaments (IFs) form an essential part of the metazoan cytoskeleton. Despite a long history of research, a proper understanding of their molecular architecture and assembly process is still lacking. IFs self-assemble from elongated dimers, which are defined by their central "rod" domain. This domain forms an α-helical coiled coil consisting of three segments called coil1A, coil1B, and coil2. It has been hypothesized that the structural plasticity of the dimer, including the unraveling of some coiled-coil regions, is essential for the assembly process. To systematically explore this possibility, we have studied six 50-residue fragments covering the entire rod domain of human vimentin, a model IF protein. After creating in silico models of these fragments, their evaluation using molecular dynamics was performed. Large differences were seen across the six fragments with respect to their structural variability during a 100 ns simulation. Next, the fragments were prepared recombinantly, whereby their correct dimerization was promoted by adding short N- or C-terminal capping motifs. The capped fragments were subjected to circular dichroism measurements at varying temperatures. The obtained melting temperatures reveal the relative stabilities of individual fragments, which correlate well with in silico results. We show that the least stable regions of vimentin rod are coil1A and the first third of coil2, while the structures of coil1B and the rest of coil2 are significantly more robust. These observations are in line with the data obtained using other experimental approaches, and contribute to a better understanding of the molecular mechanisms driving IF assembly.