Estimating the Cholesterol Affinity of Integral Membrane Proteins from Experimental Data

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Integral plasma membrane proteins interact with cholesterol, influencing their function.
• Previous estimations of cholesterol affinities relied solely on molecular computation, lacking experimental validation.

Purpose of the Study:

• To develop a mathematical model for experimentally determining sterol affinity constants and stoichiometries of integral plasma membrane proteins.
• To analyze cholesterol binding characteristics of various membrane proteins.

Main Methods:

• Developed a mathematical model to analyze published isotherms of protein activity versus membrane cholesterol concentration.
• Extracted sterol affinity constants and stoichiometries by fitting the model to experimental data.
• Utilized published values for phospholipid sterol association constants and stoichiometries.

Main Results:

• The model successfully matched experimental data, revealing sigmoidal binding curves with lagged thresholds due to phospholipid competition.
• Three oligomeric transporters (Kir3.4*, Kir2, GAT) bind cholesterol without cooperativity, exhibiting lower affinities than phospholipids.
• The BK channel, nicotinic acetylcholine receptor, and a Kir3.4* mutant bind cholesterol cooperatively with higher affinities.

Conclusions:

• The developed model provides experimental validation for cholesterol-protein interactions.
• Protein sensitivity to in vivo cholesterol variations depends on their binding avidity.
• The method is broadly applicable to integral proteins and other ligands in lipid bilayers.