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Substrate-induced activation of a trapped IMC-mediated protein folding intermediate

M Inouye1, X Fu, U Shinde

  • 1Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.

Nature Structural Biology
|March 29, 2001
PubMed

Insights

Protein folding intermediates can gain catalytic activity. A crosslinked intermediate conformer (CLIC) of subtilisin was activated to show substrate specificity distinct from the fully folded enzyme.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Protein folding intermediates are transient states during protein folding.
  • Their physiological relevance in folding kinetics and catalytic function remains debated.
  • Intramolecular chaperones (IMC) can stabilize partially folded protein states.

Purpose of the Study:

  • To investigate the catalytic potential of a trapped protein folding intermediate.
  • To characterize the structural and functional properties of this intermediate.
  • To explore the substrate specificity of the activated intermediate.

Main Methods:

  • Utilizing an intramolecular chaperone (IMC) to create a disulfide-linked crosslinked intermediate conformer (CLIC) of subtilisin.
  • Inducing catalytic activity in CLIC by incubation with small peptide substrates.
  • Comparing the structural and catalytic properties of the activated CLIC (A-CLIC) with the fully folded enzyme.

Main Results:

  • A stable, partially folded crosslinked intermediate conformer (CLIC) was successfully trapped.
  • CLIC was induced into a catalytically active form (A-CLIC) upon substrate binding.
  • A-CLIC exhibited distinct catalytic properties and lacked endopeptidase activity on large protein substrates compared to fully folded subtilisin.
  • A-CLIC demonstrated substrate specificity different from the native enzyme.

Conclusions:

  • Partially folded protein intermediates, stabilized by disulfide bonds, can acquire catalytic activity.
  • These intermediates can possess unique substrate specificities, differing from the native enzyme.
  • Protein folding intermediates may play active roles in catalytic reactions, challenging traditional views of enzyme function.

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