Detergent Exchange from Lipid Nanoparticles into Detergent Micelles Unlocks a Tool for Biochemical and Kinetic...

Bacterial membrane proteins make up ∼ 30% of the prokaryotic genome and play key roles in infection and virulence. Membrane protein chemistry has advanced in recent years, including purification strategies that mimic nativelike lipid environments, such as lipid nanoparticles, amphipols, and nanodiscs. The use of styrene maleic acid copolymers (SMALPs) to form a lipid nanoparticle has become increasingly common in membrane protein purification, especially for proteins which are not amenable to detergent extraction from the cellular membrane fraction. Yet, for some biochemical and biophysical methods, it is preferable to use detergent-solubilized protein. Here we show a general exchange screening method to transfer membrane proteins from lipid nanoparticles to detergent micelles while retaining protein fold, homogeneity, and function. Conditions were first optimized for copolymer dispersion and recovery into detergents, and analytical methods were employed to assess activity and quality of detergent-solubilized proteins. Thirteen protein targets were purified in copolymer based on a 16-polymer screen. This selection was followed by an eight-detergent screen in the presence of calcium and magnesium ions for optimal dissolution of the nanoparticle, producing detergent-stabilized protein. In all membrane proteins assessed, homogeneity and folding were retained from the initial purification in lipid nanoparticles through a detergent-exchange protocol. For membrane enzymes that have proven to be experimentally intractable when detergent solubilized, we were able to observe catalytic activity using the detergent-exchanged material. The use of this protocol to purify membrane proteins provides greater versatility for biochemical and kinetic characterization than was previously accessible.