Quantifying protein interface footprinting by hydroxyl radical oxidation and molecular dynamics simulation:...

Area of Science:

• Biochemistry
• Structural Biology
• Chemical Biology

Background:

• Biomolecular surface mapping is crucial for understanding protein interactions when high-resolution 3D structures are unobtainable.
• Hydroxyl radical footprinting offers enhanced resolution over traditional chemical methods for characterizing these interactions.

Purpose of the Study:

• To apply hydroxyl radical footprinting using laser-induced hydrogen peroxide photodissociation to map protein-protein interfaces.
• To characterize the homodimeric interface of galectin-1 using this advanced footprinting technique.

Main Methods:

• Hydroxyl radicals were generated via laser-induced photodissociation of hydrogen peroxide on a nanosecond timescale.
• Mass spectrometry data analysis was performed using modified ByOnic software to manage extensive oxidation modifications.
• Quantification of oxidation levels was achieved using spectral intensities on a per-residue basis.

Main Results:

• A direct correlation was established between side-chain solvent accessibility and the observed level of oxidation.
• The method enabled prediction of oxidation levels based on a protein's 3D structure.
• Molecular dynamics simulations refined the accuracy of solvent accessibility calculations, enhancing predictive precision.

Conclusions:

• Hydroxyl radical footprinting provides a precise method for biomolecular surface mapping and characterizing protein interfaces.
• The established relationship between oxidation and solvent accessibility facilitates structural predictions for protein complexes.
• This technique offers a valuable alternative for studying protein-protein and protein-ligand interactions.