Expression and purification of large nebulin fragments and their interaction with actin

cDNA clones encoding mouse skeletal muscle nebulin were expressed in Escherichia coli as thioredoxin fusion proteins and purified in the presence of 6 M urea. These fragments, called 7a and 8c, contain 28 and 19 of the weakly repeating similar to 35-residue nebulin modules, respectively. The nebulin fragments are soluble at extremely high pH, but aggregate when dialyzed to neutral pH, as assayed by centrifugation at 16,000 x g. However, when mixed with varying,amounts of G-actin at pH 12 and then dialyzed to neutral pH, the nebulin fragments are solubilized in a concentration-dependent manner, remaining in the supernatant along with the monomeric actin. These results show that interaction with G-actin allows the separation of insoluble nebulin aggregates from soluble actin-nebulin complexes by centrifugation. We used this property to assay the incorporation of nebulin fragments into preformed actin filaments. Varying amounts of aggregated nebulin were mixed with a constant amount of F-actin at pH 7.0. The nebulin aggregates were pelleted by centrifugation at 5200 x g, whereas the actin filaments, including incorporated nebulin fragments, remained in the supernatant. Using this assay, we found that nebulin fragments 7a and 8c bound to actin filaments with high affinity. Immunofluorescence and electron microscopy of the actin-nebulin complexes verified that the nebulin fragments were reorganized from punctate aggregates to a filamentous form upon interaction with F-actin. in addition, we found that fragment 7a binds to F-actin with a stoichiometry of one nebulin module per actin monomer, the same stoichiometry we found in vivo. In contrast, 8c binds to F-actin with a stoichiometry of one module per two actin monomers. These data indicate that 7a can be incorporated into actin filaments to the same extent found in vivo, and suggest that shorter fragments may not bind actin filaments in the same way as the native nebulin molecule.