Studies on the structure and function of a biomotor dynein

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Dynein is a very huge motor protein in contrast to myosin and kinesin. There are two isoforms of dyneins; axonemal and cytoplasmic ones. However, dyneins so far cloned have four P-loop (ATP-binding) motives in the midregion of the molecules. In the present work, I have determined the true ATPase activity site of outer arm dynein from sea urchin sperm axonemes by the recombinant protein expression.In an expression of a series of deleted mutants in Baculo virus system, products were not extracted under the mild condition possibly because of an abortive folding of expressed mutants. To get soluble mutants, we have tried the dual expression of mutants and the other protein component contained in outer arm dynein. In situ dynein (heavy chain of outer arm dynein) associates with intermediate chain (IC1). Thus, we expressed simultaneously a series of mutants and IC1 in Baculo virus system.ICI was flagged with 6xHis. Taking the in situ binding of dynein heavy chain and IC1 into consideration, … More mutants could be bound to the Ni-affinity resin via the interaction of them with IC1-6xHis. I confirmed that mutants were bound to the resin, although the percentages of bound and unbound ones were very low.Mutant ranged in the four P-Loop motives showed an ATPase activity, though to greatly Lesser extents than in situ dynein. So, I prepared a series of smaller mutants containing only one P-Loop motif in the molecule to identify the ATPase site of dynein heavy chain and the significance of four P-Loop motives. Dual expression of them and ICI-6xIlis and ATPase assay demonstrated that only the mutant containing N-terminal first P-Loop (PI) showed ATPase activity. The other P-loop motif itself didn't show any ATPase activity.From the present work, the first P-loop motif of dynein molecule seems responsible for ATPase activity of dynein. Although the solubility of mutants was improved by the dual expression, Low specific activity of mutants suggest the occurrence of still abortive folding of them. Less