Membrane Protein Solubilization

How Do Detergents Solubilize Membrane Proteins?

Detergents are widely used in biochemistry, cell biology and molecular biology. Common applications include cell lysis, solubilization of membrane proteins and lipids, protein crystallization, and reduction of background staining in blotting experiments.

Even though studying membrane proteins is a major challenge in protein biochemistry, they remain an important area of study due to their significant biological and pharmacological relevance. Understanding the structure and function of membrane proteins requires their careful isolation in the native form in a highly purified state. Since micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers, their use as solubilizing agents is essential for functional and structural investigations.

Biological membranes consist of phospholipids that contain two hydrophobic groups connected to a polar head. This molecular architecture allows phospholipids to assemble as lipid bilayers in which the hydrophobic chains face each other while the polar head groups face outward to the aqueous milieu (Figure 1). Proteins and lipids are embedded in this bilayer forming the fluid mosaic model (Figure 2) which was first proposed by Singer and Nicolson in 1972.

Proteins are held in the lipid bilayer by hydrophobic interactions between the lipid tails and hydrophobic protein domains.¹ These integral membrane proteins (IMPs) (Figure 2) are not soluble in aqueous solutions as they aggregate to protect their hydrophobic domains, but are soluble in detergent solutions as micelles formed by detergents are analogous to the bilayers of the biological membranes.² Proteins are incorporated into these micelles via hydrophobic interactions. Hydrophobic regions of membrane proteins, normally embedded in the membrane lipid bilayer, are now surrounded by a layer of detergent molecules and the hydrophilic regions are exposed to the aqueous medium. This keeps the membrane proteins in solution. Complete removal of detergent could result in aggregation due to the clustering of hydrophobic regions and, hence, may cause precipitation of membrane proteins.³

Membrane solubilization by detergents can be described as a three stage process where the detergent-lipid-protein ratio is an important factor (Figure 3).⁴

When low concentrations of a detergent are added to biological membranes (a), the detergent monomers (shown in red with single tails) perturb the membrane structurally by partitioning into the lipid bilayer (b). At concentrations equal to, or higher than the detergent’s CMC, the lipid bilayer becomes saturated with detergent molecules and breaks apart generating lipid-protein-detergent mixed micelles (c). 8 A detergent/protein ratio of around 1-2 (w/w) is believed to be sufficient to solubilize IMPs to form lipid-protein-detergent mixed micelles.⁵ A further increase of detergent concentration causes progressive delipidation of the lipid-protein-detergent mixed micelles. This leads to the formation of lipid/detergent and protein/detergent mixed micelles (d). 8 A solubilized IMP in a complex with a bound detergent is called a protein-detergent complex, PDC.⁶ Typically, a detergent/protein ratio of around 10 (w/w) or higher will lead to complete delipidation.⁷