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.

Preliminary Evidence for Binding of Myosin to a Portion of Twitchin

Mario Valenzuela and Guy Benian

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 
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.