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729c

Studying the Structural Properties of Nesprin-1α By Using Engineered Protein Fragments

Zhixia Zhong1, Siwei Chang1, Katherine Wilson2, and Kris Noel Dahl1. (1) Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213, (2) Johns Hopkins University, 725 N. Wolfe St., G10, Baltimore, MD 21205

Nesprins, encoded by three genes in humans, play key roles in mechanically linking the cytoskeleton to lamin intermediate filaments in the nucleus. One particular isoform, nesprin-1α (120 kDa), is an integral membrane protein of the inner nuclear membrane that binds directly to A-type lamins and emerin. Nesprin-1α plays a role in the pathogenesis of Emery-Dreifuss muscular dystrophy (EDMD), and heterozygous missense mutations in nesprin-1α lead to EDMD.Structurally, nesprin-1α has six spectrin like repeats (SLR) and a central rod domain, comprising 25% of its length. The SLR domains in nesprin-1α are weakly homologous to α- and β-spectrin (21-28%), but the structural properties of nesprin-1α, itself, were untested and the structural contributions of of the rod domain were unclear. Thermodynamic properties of other spectrin superfamily proteins involve cooperative coupling folding and unfolding of tandem three-helix bundles (SR domains). We hypothesized that the rod domain of nesprin-1α might confer unique molecular thermodynamics compared to other SR domain proteins. To test this model, we tested the thermodynamic stability of several nesprin-1α fragments including an engineered fragment of the full-length nesprin-1α without the rod domain. We measured the secondary structure and thermal unfolding properties of the constructed protein fragments of nesprin-1α fragments using circular dichroism. Despite the weak homology with spectrin, the predicted SLR domains in nesprin-1α have thermodynamic properties of typical SR domains based on their helicity, mean transition temperatures (Tm), ellipticity ratio Θ222208, multiple transition stages and cooperative coupling unfolding. However, the protein fragments that included the rod domain were highly helical with a higher Tm than corresponding fragments lacking the rod domain. Thus, the rod domain overstabilizes the SLR domains by increasing the cooperative folding and by increasing overall helicity and Tm of the molecule. We also tested these protein fragments to look for in vitro binding to emerin. We found that the linker region is not necessary for protein binding, but our results suggest that the larger protein structure may be required for binding studies due to the cooperative folding of these SLR segments. Currently we are examining these engineered nesprin-1α fragments expressed in Hela cell to test the cell effects of the rod domain and SR domains. A combination of in vitro and cellular studies examining protein structure and interaction may lead to a better understanding of nuclear lamina organization as well as elucidate the mechanism of EDMD.