N-Ethylmaleimide-modified Hsp70 inhibits protein folding.
Hsp70 molecular chaperones facilitate protein folding and translocation by binding to hydrophobic regions of nascent or unfolded proteins, thereby preventing their aggregation. N-Ethylmaleimide (NEM) inhibits the ATPase and protein translocation-stimulating activities of the yeast Hsp70 Ssa1p by modifying its three cysteine residues, which are located in its ATPase domain. NEM alters the conformation of Ssa1p and disrupts the coupling between its nucleotide- and polypeptide-binding domains. Ssa1p and the yeast DnaJ homolog Ydj1p constitute a protein folding machinery of the yeast cytosol. Using firefly luciferase as a model protein to study chaperone-dependent protein refolding, we have found that NEM also inhibits the protein folding activity of Ssa1p. Interestingly, the NEM-modified protein (NEM-Ssa1p) is a potent inhibitor of protein folding. NEM-Ssa1p can prevent the aggregation of luciferase and stimulate the ATPase activity of Ssa1p suggesting that it acts as an inhibitor by binding to nonnative forms of luciferase and by competing with them for the polypeptide binding site of Ssa1p. NEM-Ssa1p inhibits Ssa1p/Ydj1p-dependent protein refolding at different stages indicating that the chaperones bind and release nonnative forms of luciferase multiple times before folding is completed.
Hermawan
A
A
Department of Anatomy and Cell Biology, State University of New York-Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, New York, 11203, USA.
Chirico
W J
WJ
D000251:Adenosine Triphosphatases; D000818:Animals; D001517:Coleoptera; D003600:Cytosol / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D018840:HSP70 Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; D008156:Luciferases; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae; D005033:Ethylmaleimide