Worm Breeder's Gazette 11(4): 45
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Twitchin was the first intracellular protein discovered to be a member of the immunoglobulin superfamily. Twitchin is the first member of a growing family intracellular muscle proteins found in diverse muscle types and animals composed of multiple copies of motif I (fibronectin type III domain-like) and motif II (immunoglobulin C2 domain-like). To date, these include, myosin light chain kinase of smooth muscle (chicken; PNAS 87, 2284, 1990), titin (striated muscle of rabbit; Nature 345, 273, 1990), C-protein and 86 kDa protein ( striated muscle of chicken; PNAS, 87, 2157, 1990), skelemins (striated mammalian; M. Price, pers. comm.) and probably projectin (insects; M. L. Pardue, pers. comm.). The similarities to motifs found in cell surface proteins engaged in adhesion or recognition suggests that motifs I and II of muscle proteins are also involved in binding-- probably binding to myosin and also binding of these proteins to themselves. One of our goals is to find out whether small numbers of these motifs from twitchin can be shown to bind to myosin in vitro. Using the pGEX-2T expression vector, we have been able to generate large quantities of motif I and the predominant triplet I, I, II in E. coli. These are expressed as fusion proteins with glutathione-S- transferase (GST) at the N terminus. The GST moiety permits affinity purification of our proteins with glutathione-agarose beads (GSH beads) . In addition, the fusion protein has a potential thrombin cleavage site between the GST portion and the motifs. Unfortunately, our attempts at thrombin cleavage have been disappointing. (We are also trying to express these motifs with ATG vectors in baculovirus- infected insect cells). Nevertheless, we surmised that the GST portion of our fusion proteins may provide a way to enrich for proteins that bind to the motifs. Clarified rabbit myosin minifilaments were incubated alone, with GST, with GST-I, or with GST- I,I,II. Incubation was at 20 C overnight, in 10 mM citrate-tris pH8, 2.9 mM MgS04, 12.5 mM KCl and 0.6 mM ATP (binding buffer). The following day, GSH beads in binding buffer were added to each of the four reactions. After incubation at room temperature with shaking ( only the tubes, not the student), the GSH beads were pelleted and separated from the supernatant. The pellets were washed 5 times with 1 ml of binding buffer. The samples were analyzed by SDS-PAGE using a 4-12% gradient gel and silver stained. Although 90-100% of GST and GST-I were found to be associated with the GSH beads, the amount of myosin in their respective supernatants was about twice that in their respective pellets. The results with these proteins were not much different from addition of GSH beads to myosin alone. In contrast, there was a parallel enrichment of myosin and GST I,I,II in the pellet fraction. This suggests that myosin was pulled-out of the supernatant by binding to the motif portion of GST-I,I,II. Soon, we will try nematode myosin. To optimize this assay (e.g. improve washing) and to determine which portion of myosin binds to the motifs, we will employ myosin proteolytic subfragments. Ultimately, we hope to work with isolated motifs and try more direct binding studies.