Programming xenon diffusion in maltose-binding protein.

Area of Science:

• Biophysics
• Biochemistry
• Structural Biology

Background:

• Protein interiors possess void spaces capable of binding small gas molecules.
• Understanding gas diffusion pathways and kinetics within proteins is a significant scientific challenge.

Purpose of the Study:

• To investigate xenon (Xe) exchange kinetics in maltose-binding protein (MBP) using computational methods and hyperpolarized xenon-129 chemical exchange saturation transfer (hyper-CEST) NMR.
• To elucidate the role of ligand binding and specific protein sites in modulating Xe exchange pathways.

Main Methods:

• Combined computational approaches with hyperpolarized xenon-129 chemical exchange saturation transfer (hyper-CEST) NMR spectroscopy.
• Utilized "Xe flooding" molecular dynamics simulations to identify and characterize Xe binding sites and pathways.
• Employed site-directed mutagenesis and 13C NMR spectroscopy to validate computational findings and assess exchange dynamics.

Main Results:

• Maltose binding induced a salt bridge formation near the Xe-binding site, significantly slowing Xe exchange and enabling hyper-CEST detection in the maltose-bound state.
• Xe dissociation was found to be faster than the dissociation of the salt bridge.
• Molecular dynamics simulations identified a secondary Xe exchange pathway through a surface hydrophobic site (V23), which was confirmed by mutagenesis studies.
• Mutations at V23 modulated Xe diffusion, demonstrating site-specific control over xenon-protein-solvent exchange.

Conclusions:

• The study demonstrates the ability to control xenon-protein-solvent exchange by targeting specific protein sites.
• The findings suggest that the hydrophobic cavity in MBP plays a role in accommodating structural changes during ligand binding and protein interactions.
• This work highlights the utility of hyper-CEST NMR and computational methods for probing gas dynamics in proteins.