title: A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state.
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 degrees 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.
authors.affiliation: Department of Biochemistry, The University of Arizona, Tucson 85721-0106, USA.
authors.forename: G J
authors.initials: GJ
authors.lastname: Lee
authors.forename: A M
authors.initials: AM
authors.lastname: Roseman
authors.forename: H R
authors.initials: HR
authors.lastname: Saibil
authors.forename: E
authors.initials: E
authors.lastname: Vierling
mesh_terms: D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002850:Chromatography, Gel; D002950:Citrate (si)-Synthase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018532:Pisum sativum / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D017421:Sequence Analysis; D013696:Temperature
chemical_list: D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D007074:Immunoglobulin G; D010446:Peptide Fragments; D010940:Plant Proteins; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D008291:Malate Dehydrogenase; D008156:Luciferases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D002950:Citrate (si)-Synthase