# Structural Biochemistry/Membrane Proteins/Methods for Studying Membrane Proteins

Studying membrane proteins is very difficult. Extracting, purifying, homogenizing or removing them from the membrane could mean serious loss of information and protein unwinding and unfolding. The best way to study protein membranes is to mimic their native environment in the cell, embedded or attached to the cell membrane.

## X-Ray Crystallography

Membrane proteins have proven to be very difficult to crystallize through X-ray crystallography mainly due to the large percentage of non-polar amino acids present within the primary structure. Traditionally, while crystallizing a membrane protein sample, scientists have tried to remove a large amount of the lipids surrounding the membrane proteins during the preparation of the sample. However, now scientists are beginning to recognize the lipids as important additives during the crystallizing process. Either lipid additions or the use of lipid cubic phases have led to an increasing number of solved membrane proteins. With the lipids attached, scientists are able to classify and figure out the different interactions between the proteins and lipids

## Lipopeptide Detergents

Traditionally, detergents have been used to extract membrance proteins using PDCs (protein-detergent complexes) for study such as X-Ray Crystallography or NMR. The effectiveness of the specific detergent used is based upon what is the desired protein. A wide variety of detergents form spherical or ellipsoid micelles which poorly imitate the cell membrane to form complexes with the protein. Membrane proteins in this detergent-solubilized state are prone to issues such as irreversible aggregation or the loss of their native structure. Being solubilized in detergent also causes constant and rapid interchanging of the bound detergent molecules with the free molecules in the solution. This causes NMR spectroscopy to be very difficult as it often requires high temperatures over an extended period of time. NMR had been limited to especially hardy proteins. Also, because of the amount of detergent molecules that bind to the protein, the structure appears disordered and is not visible in an electron density map.
This issue with detergents led scientists to believe that membrane proteins were delicate relative to other proteins. When in the cell membrance, however, integral proteins are stabilized by multiple factors including the shielding of the hydrophobic portions of the protein and lateral pressure exerted by the lipid bilayer. These factors are not present in the detergent-solubilized state. Also, the introduction of artifacts from the detergent may appear. That causes the membrane proteins to be more susceptible to their loss of native structure which also results in the deactivation of the protein. The effectiveness of the detergents used is based on the particular protein and the detergent used. A poor detergentwill serve as a poor substitute for the lipid bilayer, causing the protein to degrade. This all interferes with the studies of the proteins. Currently, Dodecyl-B-D-maltopyranoside (DDM) is currently the most effective detergents for maintaining protein stability. This is due to the formation of large micelles by the detergent which better imitate the lipid bilayer.
PDCs are solubilized membrane proteins which exist in a complex with a detergent. This particle exists in solution with free detergent monomers and detergent micelles. There is a constant exchange of bound detergent molecules and the free molecules in the solution. The amount of detergent bound to the protein of interest can range from 30-70% of the mass of the PDC, amounting to hundreds of detergent molecules bound to the protein. These molecules also do no arrange themselves with their hydrophobic chains along the hydrophobic portion of the protein. They, instead, arrange themselves to be normal to the protein and appear disordered in X-ray crystal structures and electron density maps.
Another detergent has been developed to more effectively replicate the membrane bilayer to stabilize the membrane proteins which allows them to be studied more effectively and while they are able to function. Lipopeptide Detergents are amphiphiles with a 25-residue peptide that forms an amphipathic α-helix with two fatty acid groups ranging between 12 and 20 carbons at the 2 and 24 positions. These chains lie along the hydrophobic surface and form a wedge shake. They are able to form small, cylindrical micelles with an interior that more closely resembles the lipid bilayer. This allows the membrane proteins to last longer against aggregation and denaturation. Also, compared to traditional detergents, LPDs are relatively small and keep a rigid exterior surface. The rigidity of the complexes favor both crystallization and NMR. X-Ray Crystallography, for example provided a resolution of 10Å with traditional detergents while LPDs have been able to provide a resolution of 1.20Å due to the highly ordered nature of LPDs.
LPDs, however, are not able to replace PDCs due to their higher cost. Currently, purification of the membrane proteins is still carried out with PDCs to extract the proteins and solubilize them. Once the proteins are extracted, LPDs are added to the solution and the mixture is concentrated by centrifugation in an ultrafiltration concentrator that allows the smaller LPDs to pass through while filtering out the larger PDC complexes. More buffer is added to the solution and the filtration process is repeated for several cycles, the result being the removal of PDCs and the solution of stabilized proteins in LPDs. This method requires the minimal use of LPDs which reduces the cost of membrane protein research.
While LPDs are a relatively new and effective tool for studying the structure of proteins, it is simply another method of studying proteins. It is not the single solution to the various issues in protein studies. It does, however, provide a new avenue to study more integral proteins that have challenged earlier methods. Lipopeptide Detergents.

## Importance of Solubilizing Environment on Membrane Proteins

Cell membranes constitute an array of different compositions. The heterogeneous environment effects membrane proteins in their structure and therefore, in their function. This heterogeneous environment makes it difficult for present methods to determine the structure of membrane proteins because the alteration of the membrane environment causes the structure to change. And although membrane proteins are significantly important because of their roles in biology, few of them have had their structures determined.
Protein-membrane interactions are greater than intra-protein interactions for smaller proteins and for proteins with out prosthetic groups. Prosthetic groups help overcome protein-membrane interactions by stabilizing the proteins transmembrane domains. For larger proteins, the intra-protein interaction is significant enough to overcome these interactions. Therefore, special attention must be given to the environment of smaller proteins when deciphering their structure. Since membrane proteins have significant conformational flexibility which may be necessary for their function under native conditions. An example of this would be the structural change that ion channels undergo when opening or closing. Both of these structures contribute to the proteins function and require the membrane to allow this change to occur by overcoming the protein-membrane interactions. There can also be conformational changes that do not contribute to the function of the protein and may well inhibit the protein's function. These changes can be caused by the same protein-membrane interactions. This gives rise to a special concern when unraveling the structure of new proteins.
This concern is whether the structure being analyzed is in fact the, or one of the functional structures of the protein. A case such as this is seen in the influenza A virus M2 protein. This protein's structure has been determined from samples in liquid crystalline lipid bilayers, samples crystallized from detergents and samples in detergent micelles. Each of the samples represents different protein structure and it's not clear which contribute to the function of the protein. Understanding which structure is relevant to the function will aid in developing pharmaceuticals that can efficiently disrupt the replication of the virus and prevent flu pandemics mutate and no longer are susceptible to previous drug therapy.
While using detergents to solubilize proteins, it is important to note that differences between lipids and detergents can influence protein structure. One difference is that monomeric lipid concentrations are many orders of magnitude lower than millimolar concentrations of monomeric detergents. In some protein crystal structures, these monomeric detergents have been seen to penetrate aqueous pores of protein domains which can compromise its functionality. Second, detergent micelles are expandable in their hydrophobic sections. This can cause helices, designed to have a hydrophobic mismatch, to induce helical tilt and cause them to be tightly packed. This can also affect protein function. Finally, the micelle surface has a greater degree of curvature when compared to a membrane surface. This change in curvature can also interfere with the surface binding of amphipathic helices. These changes all involve the environment of the membrane protein. These interactions can have significant effect on protein structure and further investigation is needed to uncover all relationships between the two.

## References

* 1 2 3 Prive, G. Lipopeptide Detergents for Membrane Protein Studies, Current Opinion in Structural Biology, Volume 19, Issue 4, Pages 379-385 August 2009
* ↑ Cross T a, Sharma M, Yi M, Zhou H-X. Influence of solubilizing environments on membrane protein structures. Trends in biochemical sciences. 2011;36(2):117–25. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3161620&tool=pmcentrez&rendertype=abstract. Accessed November 9, 2012.