Maize shsp18-9 (uwo 11) has been previously shown to undergo strong induction during heat shock (Goping et al., Plant Mol. Biol.16:699-711, 1991). It also shows developmentally modulated expression at several stages in the growing anthers and spikelets (Bouchard et al., Maydica 38:135-144, 1993) as well as induction by insult with toxic heavy metals (Yang and Walden, MNL 71:55, 1997). Construction of a plasmid encoding fusion protein TRCHIS18-9 and preparation and affinity-purification of the product under denaturing conditions was as described in Greyson et al. (Dev. Genetics 18:244-253, 1996). Purified TRCHIS18-9 was then dialyzed into 50mM Sodium Acetate buffer, pH 4.5, aliquoted, and frozen until use.
We examined the proteinís capacity for dodecamer formation using pore gradient Native-PAGE electrophoresis at temperatures ranging from 4 C and 43 C. Under all conditions, all of the fusion protein ran as a defined band with an estimated molecular weight of 250 kDa relative to native protein standards, consistent with formation of a single dodecameric oligomer. A characteristic example of this oligomer alongside Native-PAGE standards is shown in Figure 1 below.
We also tested chaperone activity of this oligomer using the substrate protein citrate synthase (CS), an enzyme which normally undergoes rapid, irreversible aggregation and denaturation upon incubation at 43 C. In our assays, a solution containing CS was incubated at denaturing temperature and samples withdrawn at regular intervals were then assayed for residual activity at 25 C. In the absence of TRCHIS18-9, CS was rendered totally inactive after 10 minutes of incubation at 43 C. Upon incubation with increasing ratios of TRCHIS18-9, however, higher and higher proportions of the CS activity were preserved. At our highest tested ratio of 15:1 of TRCHIS18-9 dodecamer to CS homodimer, nearly full CS activity was found in samples even after 50 minutes at 43 C, the longest incubation period tested.
These results suggest that maize sHSP18-9 may provide molecular chaperone
activity under a variety of stress and developmental conditions where it
is expressed. We are currently performing additional experiments to investigate
the role of conserved amino acids and structural motifs in oligomer formation
and chaperone function.
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