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Visualizing an Allosteric Intermediate Using CuAAC Stabilization of an NMR Mixed Labeled Dimer.

Paul J Sapienza1, Michelle M Currie1, Noah M Lancaster1

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Summary
This summary is machine-generated.

Researchers developed a method to stabilize and isolate the elusive singly bound (lig1) intermediate of allosteric homodimers, like chorismate mutase. This breakthrough enables detailed NMR studies of allostery and protein interactions.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Homodimeric enzymes are abundant and crucial for studying allostery.
  • The asymmetric singly bound (lig1) intermediate is key to understanding allosteric mechanisms but is difficult to isolate.
  • Allostery involves how ligand binding at one site affects binding at another.

Purpose of the Study:

  • To develop a method for obtaining and stabilizing the lig1 intermediate of allosteric homodimers.
  • To enable detailed structural and functional investigations of allosteric mechanisms.
  • To facilitate protein linkage applications in structural biology.

Main Methods:

  • Utilizing copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to link subunits of chorismate mutase.
  • Incorporating unnatural amino acids to create mixed isotopically labeled dimers.
  • Developing protocols to overcome CuAAC pitfalls for high-yield protein production.
  • Purifying the mixed-labeled dimer to obtain the active lig1 intermediate.

Main Results:

  • Successfully generated milligram quantities of the lig1 intermediate of chorismate mutase.
  • Produced a highly purified, fully intact, and active mixed-labeled dimer.
  • Overcame challenges associated with CuAAC for protein conjugation.
  • Provided the first structural glimpse of the lig1 intermediate.

Conclusions:

  • The developed method allows for the isolation of the elusive lig1 intermediate of allosteric homodimers.
  • This facilitates NMR-based investigations into the mechanisms of allostery.
  • The technique is applicable to other structural studies requiring specific protein linkage.