Precursor Reagent Hydrophobicity Affects Membrane Protein Footprinting

Area of Science:

• Biochemistry
• Structural Biology
• Chemical Biology

Background:

• Membrane proteins (MPs) are vital for cellular functions and drug targets.
• Structural characterization of MPs is challenging due to their hydrophobic nature.
• Existing water-soluble reagents limit the study of hydrophobic MP regions.

Purpose of the Study:

• To evaluate MS-based photoactivated iodine reagents with varying hydrophobicities for MP structural characterization.
• To improve reagent access to hydrophobic MP domains using tip sonication.
• To provide residue-level structural information for MPs.

Main Methods:

• Utilized a series of MS-based photoactivated iodine reagents with different hydrophobicities.
• Employed tip sonication to enhance reagent diffusion into micelles and MP hydrophobic cores.
• Analyzed modification extent in hydrophilic and hydrophobic domains using proteolysis and LC-MS/MS.
• Studied the model MP, vitamin K epoxide reductase (VKOR).

Main Results:

• Demonstrated that reagent hydrophobicity influences preferential labeling sites.
• Obtained residue-level structural information from both hydrophilic and hydrophobic MP domains.
• Successfully characterized the model MP, vitamin K epoxide reductase (VKOR).

Conclusions:

• Developed a method to overcome challenges in studying hydrophobic membrane proteins.
• The findings guide the development of chemical probes for MPs.
• Promotes high-throughput, information-rich characterization of MPs for biochemistry and drug discovery.