Substrate-induced activation of a trapped IMC-mediated protein folding intermediate.
While several unfolded proteins acquire native structures through distinct folding intermediates, the physiological relevance and importance of such states in the folding kinetics remain controversial. The intramolecular chaperone (IMC) of subtilisin was used to trap a partially folded, stable crosslinked intermediate conformer (CLIC) through a disulfide bond between mutated IMC and subtilisin. The trapped CLIC contains non-native interactions. Here we show that CLIC can be induced into a catalytically active form by incubating it with small peptide substrates. The structure and catalytic properties of the activated crosslinked intermediate conformer (A-CLIC) differ from those of the fully folded enzyme in that A-CLIC lacks any endopeptidase activity toward a large protein substrate. Our results show that a disulfide-linked partially folded protein can be induced to acquire catalytic activity with a substrate specificity that is different from completely folded subtilisin. These results also suggest that protein folding intermediates may also participate in catalytic reactions.
Inouye
M
M
Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
Fu
X
X
Shinde
U
U
D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation; D008715:Methionine / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020673:Protein Renaturation; D012694:Serine / Q000378:metabolism; D020860:Subtilisin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D018832:Molecular Chaperones; D011498:Protein Precursors; D012694:Serine; D008715:Methionine; D020860:Subtilisin; D003545:Cysteine