WOOSTER, OHIO
The College of Wooster
Maize small heat shock protein 18-9 exhibits characteristics of a molecular chaperone in vitro. --Buss, J, Tsacoumangos, AM, Pett, V, Bouchard, RA Studies of several plant small heat shock proteins have indicated that they exhibit certain properties of molecular chaperones in vitro. Two key aspects have been well characterized for the Pisum sativum HSP18.1 (Lee and Vierling, EMBO J. 16:659-971, 1997). One is the formation of dodecameric oligomers under native conditions. The other is the capacity to prevent the formation of denatured aggregates by thermally-sensitive substrate proteins at temperatures where irreversible denaturation would otherwise occur. We now report initial results indicating the presence of both these activities in a bacterially expressed fusion protein comprising a 6-histidine affinity leader peptide and the product encoded by a member of the maize shsp gene family.

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.

Figure 1.

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upon incubation at 43 C. In our assays, a solution containing CS was incubated at denaturing temperature and samples withdrawn at regular intervals were then assayed for residual activity at 25 C. In the absence of TRCHIS18-9, CS was rendered totally inactive after 10 minutes of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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