SITE-DIRECTED MUTAGENESIS OF NONMUSCLE MYOSIN HEAVY CHAINS

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We have succeeded in expressing mg quantities of the head fragment of the chicken brain nonmuscle myosin heavy chain. This fragment which begins at the amino terminal residue and extends into the myosin tail is approximately 110,000 kDa as judged by its Mr in sodium dodecyl sulfate- polyacrylamide gel electrophoresis. We expressed this fragment of the brain myosin heavy chain using the baculovirus expression system. The myosin heavy chain could be seen as a prominent band in extracts of the virally infected army worm intestinal cells, and was purified by molecular sieve chromatography. Its identity was confirmed by Western blotting using a monoclonal antibody known to recognize an epitope in the head region of the myosin molecule. It was found to contain approximately 10% of the expected ATPase activity and this poor activity was attributed to misfolding of the nascent myosin heavy chain. Therefore, we are presently engineering expression of the 17 kDa myosin light chain using the baculovirus expression system and plan to coexpress both heavy and light chain in an effort to obtain a myosin fragment with full, albeit unregulated, myosin ATPase activity. Future experiments will be directed at mutating the head region of the myosin molecule in an effort to identify the various amino acid residues that contribute to myosin's biological activity.