A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state

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Abstract
The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde‐3‐phosphate dehydrogenase heated to 45°C. Under conditions in which HSP18.1 prevented aggregation of substrates, size‐exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes. SDS–PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non‐native substrates compared with other known molecular chaperones. Photoincorporation of the hydrophobic probe 1,1′‐bi(4‐anilino)naphthalene‐5,5′‐disulfonic acid (bis‐ANS) into a conserved C‐terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis‐ANS incorporates proximal to substrate binding regions and that substrate–HSP18.1 interactions are hydrophobic. We also show that heat‐denatured firefly luciferase bound to HSP18.1, in contrast to heat‐aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP‐dependent process. These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.