Membrane proteins play a key role in cellular processes including transport of molecules, signal transduction, utilization of energy and maintenance of cell and tissue structures. It has been determined by genome sequencing that around 30% of genes encode membrane proteins. Furthermore, they are pharmacologically significant as 50% of the current drugs target the membrane proteins. It is therefore of utmost importance to isolate membrane receptor complexes in functional active form for functional and structural studies, crystallization etc. However in spite of their significance, knowledge of structure and function of membrane proteins is lagging behind soluble proteins due to hurdles like low abundance and their isolation in native form from biological membrane. The obstacle of low abundance of membrane protein can be overcome with heterologous expression of these proteins and employing techniques similar to expression of soluble or cytosol proteins. Isolation of membrane proteins from biological membrane is carried out by solubilization and this process needs fine-tuning in order to purify functionally active membrane receptors complexes.
What is solubilization?
Solubilization of membrane proteins is a process in which buffered detergent solution is used to disrupt protein-lipid and lipid- lipid interactions of the biological membranes. The dissociation is controlled and results in formation of small lipid-protein or detergent-protein clusters that are soluble in aqueous solution. Since every membrane protein/receptor complex is unique, there cannot be one general protocol to functionally solubilize the receptor. However, due to recent advances and existing knowledge, key general critical factors can be outlined which a scientist must analyze/look before isolating a membrane protein.
Critical factors for functional solubilization of membrane proteins
Choice of detergent
Detergents are amphipathic molecules with a polar head and a hydrophobic tail. They display unique property of forming spherical micellar structures in aqueous solution where the hydrophobic group is buried in micelle and the polar group is in contact with aqueous solution. They are classified on the basis of charge on polar group into anionic (e.g. SDS), Cationic (e.g. CTAB), zwitterionic (e.g. CHAPS) and non-ionic (e.g. Triton X-100, polyoxyethylene). Ionic detergents such as SDS are effective in membrane solubilization but they mostly denature the protein. Detergents belonging nonionic and zwitterionic class are more suitable for membrane protein extraction as they are found to solubilize the membrane proteins while retaining their function. For example, CHAPS a mild non-denaturing zwitterionic detergent derived from naturally occurring bile salts is a popular detergent in membrane biochemistry as combines useful features of both bile salt hydrophobic group and the N-alkyl sulfobetaine type polar group. Triton X-100, a non-ionic detergent is also widely used for solubilization of membrane proteins.
Critical Micelle concentration of detergents
Critical micelle concentration (cmc) of the detergent is the concentration of detergent at which the detergent molecules self associate to for thermodynamically stable non- covalent aggregates where the non-polar group is displaced in the core and the polar group is aligned out in contact with aqueous solution to form spherical micellar structures. The CMC of a detergent varies with conditions such as pH, ionic strength, temperature and presence of proteins. CMC of a detergent is critical for membrane protein solubilization since at this concentration the detergent starts to accumulate in the biological membrane and therefore in general the concentration of detergent to solubilize membrane protein should be above the CMC of a detergent.
Detergent-Lipid-protein ratios
Membrane solubilization by detergents is complex and the ratio of detergent-lipid-protein indicates successful solubilization. Different stages of solubilization are obtained depending upon detergent –protein ratio. At lower concentration, the detergent molecules simply bind the membrane without disrupting it. As the concentration of detergent is increased, the membrane bilayer is disrupted leading to cell lysis and formation of lipid-protein-detergent micelles. Further increase in detergent concentration leads to formation of heterogenous complexes of detergent, lipid and protein with progressive delipidation of lipid-protein-detergent mixed miscelles and formation of lipid/detergent and protein/detergent mixed micelles. As the concentration of detergent is reached saturation stage there is no further solubilization of lipid or protein. However these concentrations are rarely used as these can destabilize the protein. It is recommended to use mild concentration of detergent which can strike a balance between maximum solubilization and yet preserve the protein/receptor function. This is achieved by trial and error basis and is specific to membrane protein that needs to be isolated.
Lipid environment
Since integral membrane protein/receptors are in contact with membrane lipid environment, it is likely and also proven that the lipid environment influences the structure and functionality of the receptor. It is also seen that some detergents differentially solubilize membrane lipids and this may result in inactivation of the receptor. It is therefore important to consider the relevance of immediate lipid environment to receptor function when choosing an appropriate detergent for solubilization.
Recent advances in membrane protein isolation indicates novel detergents such as glucose neopentyl glycol (GNG) and maltose neopentyl glycol (MNG) improve protein stability without compromising solubilizing properties.
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