A covalently trapped folding intermediate of subtilisin E: spontaneous dimerization of a prosubtilisin E Ser49Cys mutant in vivo and its autoprocessing in vitro.
The propeptide of subtilisin E (the N-terminal 77 amino acid extension) is required for the proper folding of the nascent mature protein and is also a potent and specific inhibitor of the active enzyme. Previous studies have demonstrated that the propeptide can renature denatured mature sequence either in cis or in trans and can be considered an intramolecular chaperone, since it is not required for activity of the mature enzyme. In this paper it is shown that a prosubtilisin-S49C mutant can be expressed in Escherichia coli either as a monomer or as a disulfide-linked dimer, (prosubtilisin-S49C)2, depending on the vector selected. Interconversion between (prosubtilisin-S49C)2 and prosubtilisin-S49C could be readily achieved by reduction and oxidation in denaturing solutions, such as guanidine hydrochloride or urea. While the monomer can undergo autoprocessing in vitro under refolding conditions, the dimer is trapped in an intermediate state which could not be processed into active enzyme. Remarkably, the autoprocessing of this trapped intermediate could be induced readily upon reduction by dithiothreitol. This disulfide-linked (prosubtilisin-S49C)2 is fairly stable, but does tend to aggregate when the ionic strength of the solution is reduced below 0.1 M. The disulfide-linked (prosubtilisin-S49C)2 has far- and near-UV CD spectra revealing the presence of both secondary and tertiary structures, respectively, similar to those of the active mature monomer. Hence this autoprocessing-competent state appears to be a "late" folding intermediate, arising after the "molten globule" state formed in the absence of the prosequence, that has no discernible tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)
Hu
Z
Z
Department of Chemistry, Rutgers University, State University of New Jersey, Newark 07102.
Zhu
X
X
Jordan
F
F
Inouye
M
M
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013381:Subtilisins / Q000737:chemistry*
D046911:Macromolecular Substances; D011498:Protein Precursors; D013381:Subtilisins