Abstract
Abstract: :
Purpose: To map protein–protein interactions between subunits within vimentin intermediate filaments and use these data to determine Intermediate Filament (IF) assembly order and structure. Methods: In vitro mutagenesis was used to introduce unique cysteine residues at positions within the vimentin polypeptide chain. Site Directed Spin Labeling (SDSL) is used to label these unique cysteines with a spin label and Electron Paramagnetic Resonance (EPR) is used to study protein structure and spin label/spin label interactions from these labeled positions at multiple stages during filament assembly. Multiple positions in the region of amino acids 170–190 (rod 1B) have been characterized at several assembly stages. Filament assembly is monitored by electron microscopy to verify assembly competence of the mutated spin labeled proteins. Results: The vast majority of spin labeled vimentin proteins are assembly competent. EPR data collected from positions labeled in regions designated rod 1B have demonstrated the alpha helical nature of this region. In a series of experiments performed with spin labeled positions within rod 1B, we have identified sites of spin/spin interaction signifying proximity between these positions. These data are consistent with an antiparallel arrangement of molecules with their rods 1B interacting (A11 alignment). Using protein subunits spin labeled at positions diagnostic for this and another key molecular arrangement, we have deduced an order of assembly for vimentin proteins forming tetramers and ultimately filaments. Conclusions: Within the range of positions we have studied, the alpha helical coiled coil nature of rod 1B has been confirmed. In addition, EPR data have identified amino acids that are adjacent to each other in the A11 arrangement and these data, in conjunction with a previously identified juxtaposed position for alignment A22 have allowed us to determine the order of assembly of vimentin proteins during in vitro assembly of vimentin IFs. Continued studies to map contact points up and down the vimentin molecule will ultimately produce a more resolved structure for vimantin IFs than is currently available.
Keywords: cytoskeleton • protein structure/function