The solubilisation and purification of an intrinsic membrane protein presents problems distinct from those encountered in purifying a conventional soluble protein. Discuss this statement.

Word count: 1860

In order to answer the question, this essay will first describe how soluble proteins are purified. It will then describe the process of solubilising an integral membrane protein specifically, and demonstrate differences between the two processes.

There are several methods for the purification of proteins in aqueous solution. Since these methods discriminate based on one characteristic that may be shared by several proteins, it is almost always necessary to use multiple methods to purify a protein from its cellular environment. First, the cell must be homogenised in order to make all the proteins within available. In theory, this presents a problem since proteins are mixed with proteases, and could be degraded. In practice vacuoles form spontaneously and quickly to mitigate this effect so it is not a problem that has to be contended with. After homogenisation, there are several chromatographic methods available to purify proteins completely. Size exclusion chromatography separates proteins based on molecular weight, as smaller proteins are retarded by the resin and so take longer to flow through. Ion exchange chromatography involves charged resin which binds charged amino acid residues. A buffer containing a high competing ion concentration is used to elute the proteins by binding them, and displacing them from the resin. The required strength of the elution buffer depends on the proteins’ isoelectric points, so by increasing the strength of the buffer, different proteins can be eluted at different times. Affinity chromatography works in a similar, but more specific way. Resin beads are labelled by antibodies which bind specific proteins. The proteins are then eluted by a buffer containing a secondary antibody which competes with the stationary antibody binding the protein, allowing the protein to elute. A high salt concentration can also be used to elute the protein by disrupting the non-covalent forces which attach it to the primary antibody. Hydrophobic interaction chromatography uses resin bound with phenyl or octyl groups to interact and separate proteins based on hydrophobicity. Using the correct method - or combination of methods - is the principal problem that needs to be solved when purifying a water soluble protein.

The important difference between non-membrane proteins and integral membrane proteins is that the latter group are not water soluble. The entire surface of the protein is held in a phospholipid bilayer by entropically driven hydrophobic forces made possible by the fact that the amino acid residues forming the part of surface which is held within the membrane, contain non-polar R-groups. This characteristic of the R-groups is what precludes the protein from being water soluble. All protein purification methods require the protein to be in aqueous solution, so insoluble proteins must be altered to allow this condition. Integral membrane proteins differ from peripheral membrane proteins, which are attached to the outside of the membrane by interactions with a particular phospholipid head group. These can be removed from the membrane relatively easily by disrupting those interactions, usually by means of a solution of high ionic strength1. Once removed from the membrane peripheral proteins are readily soluble since they have a hydrophilic surface. Anchored membrane proteins are also a different classification. They have a membrane spanning helix (usually an alpha-helix) which attaches them to the membrane, but the surface of the protein is still hydrophilic. These can be solubilised by cleaving off the membrane spanning domain with a targeted protease such as trypsin2. Proteolysis cannot be used to solubilise integral membrane proteins, since by definition, removing all membrane spanning regions on such a protein would destroy it.

Figure 1 – membrane proteins. Shown in green is an anchored membrane protein, in blue is an integral membrane protein and in red a peripheral membrane protein.

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Figure 1 – membrane proteins. Shown in green is an anchored membrane protein, in blue is an integral membrane protein and in red a peripheral membrane protein.

In order to remove an integral membrane protein from its environment, detergents must be used. Described broadly, a detergent is a hydrophobic organic molecule with a hydrophilic “head group”. In solution, these molecules aggregate to form micelles, a sphere in which the hydrophobic tails are on the inside while the head groups face outwards into the aqueous solvent. At concentrations high enough to form micelles (the critical micelle concentration (CMC))3, detergents can solubilise proteins. The hydrophobic constituent of the detergent (often an alkyl chain) interacts with the hydrophobic surface of the protein, forming a coat around it with the hydrophilic heads facing outwards. This forms a temporary hydrophilic surface around the protein, driving it out of the membrane and into aqueous solution. While detergents all obey the above description, there are several important variations in both the hydrophobic tail length and composition, and the properties of the hydrophilic head group which all make it important to select the right detergent for the right protein.

Figure 2 - a membrane protein solubilised by detergent molecules.

Detergents can be split into three types, according to the characteristics of the head group which can be either ionic, non-ionic or zwitterionic.

Ionic (harsh) detergents in turn can be split into two types – anionic or cationic - depending on the charge of the ion which is covalently attached to the hydrophobic group. As the name suggests, they have formally charged head groups. Anionic detergents are the most common, and often take the form of a sodium salt. This class of detergents tends to denature the protein by disrupting non-covalent tertiary and quaternary structure interactions as well as it disrupts protein-lipid interactions, because of its very strong solubilising effect. However this disadvantage is not ubiquitous, nor does it vary significantly between anionic and cationic detergents. Rather, it varies between individual detergents of both groups. For example, both sodium dodecyl sulphate (SDS) and sodium cholate are anionic, since the sodium dissociates in solution leaving a negatively charged head group (sulphate and carboxyl groups respectively). Sodium cholate is commonly used to solubilise proteins for purification4, whereas SDS has a denaturing effect that precludes its use for this purpose but makes it very useful for preparing proteins for gel electropheresis.

Non-ionic (mild) detergents have no formal charge, but are polar and interact with water by hydrogen bonding. They are the most common class of detergents used in purification, though not necessarily of integral membrane proteins. While they disrupt the protein-lipid forces holding the protein in the membrane, they do not disrupt tertiary and quaternary structure interactions, and thus leave the protein in its native state. The disadvantage of using a non-ionic detergent is that they are less powerful. Non-ionic detergents are most commonly used to solubilise anchored membrane proteins in instances when proteolytic release might alter the protein’s conformation or activity. Often in experiments both methods will be used to test for any difference. Since the helical anchor will only amount to a molecular weight around 2 kDa, a weak, non-ionic detergent such as butanol is almost always sufficient5. Integral membrane proteins are much larger, such as rhodopsin (over 25 kDa6), and require a stronger detergent to solubilise them.

Zwitterionic detergents have an internal salt. Despite having no overall charge, they still sometimes display the same drawbacks as ionic detergents in terms of denaturing proteins. However unlike non-ionic detergents, they are powerful enough to solubilise large proteins. CHAPS and BIG-CHAPS are zwitterionic detergents that are often used to solubilise integral proteins.

After the classification based on head groups, detergents can vary in terms of their hydrophobic tails. Tails are generally either alkyl groups, or sterols, although some other functional groups can be present in the chain. For alkyl detergents, chain length is an important variation which affects the efficacy of the detergent in a way specific to the protein. This must be determined experimentally, and individually, although planar sterols interact strongly with amino acid residues containing aromatic groups and a high proportion of such residues may indicate that a sterol-based detergent is needed. Indeed, although there are general rules regarding head groups, the way in which any detergent interacts with a protein is individual and must be determined experimentally. Proteins folded by weaker interactions must be treated more delicately, as the risk of denaturation is increased.

In order to carry out purification, detergent must be present above the critical micelle concentration throughout. Weak, non-ionic detergents can be used for this, since the protein has already been removed from the membrane and the detergent’s only function is to prevent the protein from precipitating out of solution. Often a small amount of lipid is added in order to preserve protein activity. The same chromatographic methods detailed in paragraph one are used, with notable caveats. Gel filtration remains a useful qualitative tool, but the fact that the detergent around it masks its exact molecular weight and precludes quantitative molecular weight assessment. Ion exchange chromatography is precluded by the presence of ionic detergents7. Hydrophobic interaction chromatography can be used in the presence of detergent, providing octyl groups on the resin are used to reach the hydrophobic surface of the protein. Affinity chromatography is almost always used, and does not present problems when used with detergent present.

After purification, the detergent must be removed in order to reconstitute the protein in its active form. Given that the detergent must be present above the critical micelle concentration in order to bind to the protein, it is intuitive that one way to remove the detergent is to reduce its concentration in solution. Since detergent molecules are always a lot smaller than the proteins they are solubilising, size exclusion is a useful principle for reducing detergent concentration. Dialysis is a method of size exclusion which involves diffusion across a semi-permeable membrane, the pores of which are too big for any protein to fit through and therefore cannot separate proteins from other proteins. Because the detergent only needs to be removed to below the critical micelle concentration rather than absolutely, diffusion without any extra energy applied is sufficient. Ion exchange chromatography can remove zwitterionic and ionic detergents, but the ionic strength of elution buffer and resin type required must be determined experimentally.

Upon the removal of the detergents, the hydrophobic protein will precipitate out of solution. In such a form, proteins are inactive. In order to restore activity they must be inserted into a simulation of their environment. If phospholipids are added to the solution, a cell membrane-like bilayer called a liposome will form. The protein will associate with this in the same way as it would a cell membrane. Phospholipids vary in terms of head groups bound to the phosphate group, and alkyl chain saturation and length. Some proteins interact with specific phospholipids as a requisite to activity, which means these phospholipids must be added. This characteristic of proteins is determined experimentally. The restoration of activity is necessary for study of a protein’s function, but not necessarily for other studies (eg. its structure.)

In conclusion, there are many problems specific to purifying integral membrane proteins. These problems all stem from the fact that detergents must be used to solubilise the protein before purification. The primary problem, therefore, is the removal of the protein from the membrane by detergent and includes selecting a detergent which is capable of solubilising the protein but is not a denaturing agent. Secondary problems are encountered in size exclusion, ion exchange and hydrophobicity chromatography when trying to purify the protein in the presence of detergent.

References

Stryer et al, Biochemistry 5th Edition, Ch 12
Ehlers et al, Proteolytic release of membrane proteins: studies on a membrane-protein-solubilizing activity in CHO cells. 1997. Retrieved online from: http://www.ncbi.nlm.nih.gov/pubmed/9228557 on 01/11/10
Maibaum et al, Micelle Formation and the Hydrophobic Effect. 2004. Retrieved online from: http://pubs.acs.org/doi/full/10.1021/jp037487t on 01/11/10
Moody et al, Sodium cholate extraction of rat liver nuclear xenobiotic-metabolizing enzymes. 1988. Retrieved online from http://www.ncbi.nlm.nih.gov/pubmed/3128299 on 01/11/10
Hawrylak and Stinson, The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis. 1988. Retrieved online from http://www.jbc.org/content/263/28/14368.short on 01/11/10
Protein Data Bank, Sensory Rhodopsin II. Retrieved online from http://www.rcsb.org/pdb/explore.do?structureId=1h68 on 01/11/10
Simpson, Purifying Proteins for Proteomics: a laboratory manual, Page 129

Biochemistry Dissertation – C72343

Essay 1- The solubilisation and purification of an intrinsic membrane protein presents problems distinct from those encountered in purifying a conventional soluble protein. Discuss this statement.

George Noble

4084285

University of Nottingham, School of Biomedical Sciences