Studying the interaction between ANS and the FF domain using fluorescence and amide ¹⁵N CEST NMR experiments

Area of Science:

• Biochemistry
• Structural Biology
• Protein Folding Dynamics

Background:

• The FF domain, a four-helix bundle from human HYPA/FBP11, is known to fold through intermediate states.
• Understanding protein folding pathways and interactions with small molecules is crucial for deciphering protein function and dysfunction.

Purpose of the Study:

• To investigate the binding interaction between 8-Anilinonaphthalene-1-sulfonic acid (ANS) and the A17G mutant of the FF domain (A17G FF).
• To determine which folding states of the A17G FF domain interact with ANS.

Main Methods:

• Utilized 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence spectroscopy to detect interactions.
• Employed amide 15N Chemical Exchange Saturation Transfer (CEST) NMR experiments at 16.4 T.
• Integrated data from traditional amide 1H-15N HSQC NMR and prior 15N CEST experiments.
• Performed a four-state analysis of the collected NMR data.

Main Results:

• ANS fluorescence enhancement indicated that ANS interacts with the A17G FF mutant.
• Amide 15N CEST experiments revealed that ANS binds exclusively to the I1 folding intermediate.
• The study characterized interactions under native conditions where folded, intermediate, and unfolded states are in equilibrium.

Conclusions:

• 8-Anilinonaphthalene-1-sulfonic acid (ANS) demonstrates specific binding to the I1 folding intermediate of the A17G FF domain.
• This finding provides insights into the molecular interactions governing protein folding pathways.
• The study highlights the utility of combining fluorescence and advanced NMR techniques for studying protein-ligand interactions in complex systems.