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

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.