Abstract
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.
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References
Eder, J., Rheinnecker, M. & Fersht, A. Biochemistry 32, 18–26 (1993).
Eder, J., Rheinnecker, M. & Fersht, A. J. Mol. Biol. 233, 293–304 (1993).
Shinde, U.P. & Inouye, M. J. Mol. Biol. 252, 25–30 (1995).
Booth, D.R. et al. Nature 385, 787–793 (1997).
Jamin, M. & Baldwin, R.L. Nature Struct. Biol. 3, 613–618 (1996).
Schultz, C.P. Nature Struct. Biol. 7, 7–10 (2000).
Hagihara, Y. & Goto, Y. In Molecular chaperones in the life cycles of proteins (eds Fink, A.L. & Goto, Y.) 1–33 (Marcel Decker, New York; 1997).
Baldwin, R.L. & Rose, G.D. Trends Biochem. Sci. 24, 26–33 (1999).
Baldwin, R.L. & Rose, G.D. Trends Biochem. Sci., 24, 77–83 (1999).
Creighton, T.E. Trends Biochem. Sci. 22, 6–10 (1997).
Ellis, R.J. Curr. Biol. 9, 352–355 (1999).
Shinde, U.P. & Inouye, M. Semin. Cell Dev. Biol. 11, 35–44 (2000).
Inouye, M. Enzyme 45, 314–321 (1991).
Ikemura, H., Takagi, H. & Inouye, M. J. Biol. Chem. 262, 7859–7864 (1987).
Shinde, U.P., Liu, J.J. & Inouye, M. Nature 389, 520–522 (1997).
Zhu, X., Ohta, Y., Jordan, F. & Inouye, M. Nature 339, 483–484 (1989).
Shinde, U.P., Fu, X. & Inouye, M. J. Biol. Chem. 274, 15615–15621 (1999).
Ruan, B., Hoskins, J. & Bryan, P.N. Biochemistry 38, 8562–8571 (1999).
Wang, L., Ruan, B., Ruvinov, S. & Bryan, P.N. Biochemistry, 37, 3165–3171 (1998).
Silen, J.L. & Agard, D.A. Nature 341, 462–464 (1989).
Baker, D., Sohl, J.L. & Agard, D.A. Nature, 356, 263–265 (1992).
Sohl, J.L., Jaswal, S.S. & Agard, D.A. Nature 395, 817–819 (1998).
Winther, J.R. & Sørensen, P. Proc. Natl. Acad. Sci. USA 88, 9330–9334 (1991).
Kobayashi, T. & Inouye, M. J. Mol. Biol. 226, 931–933 (1992).
Gallagher, T., Gilliland, G., Wang, L. & Bryan, P.N. Structure 3, 907–914 (1995).
Jain, S.C., Shinde U.P., Li, Y., Inouye M. & Berman, H.M. J. Mol. Biol. 284, 137–144 (1998).
Fu, X., Inouye, M. & Shinde, U.P. J. Biol. Chem. 275, 16871–16878 (2000).
Panyim, S. & Chalkley, R. Arch. Biochem. Biophys. 130, 337–346 (1969).
Greenfield, N.J. Anal. Biochem. 235, 1–10 (1996).
Brahms, S. & Brahms, J. J Mol Biol, 138, 149–178 (1980).
Acknowledgements
The authors are grateful to K. Madura, N. Greenfield, O. Mirochnitchenko and S. Phadtare for their helpful discussions.
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Inouye, M., Fu, X. & Shinde, U. Substrate-induced activation of a trapped IMC-mediated protein folding intermediate. Nat Struct Mol Biol 8, 321–325 (2001). https://doi.org/10.1038/86194
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DOI: https://doi.org/10.1038/86194
