Isolation Methods In Membrane Proteins

Isolation methods in membrane proteins

Introduction

For membrane proteins there is no universal method of insulation and purification, since it is the force with which membrane lipids that determine what procedure follow. While some can only be extracted with high ionic force solutions (NaCl 1m) others need organic detergents and solvents for separation, a factor that indicates how intimate is the protein-lipid union. 

The important role that these proteins play in the physiology of cells, and therefore, of organisms causes strong interest in molecular biology, so the study and characterization of their three -dimensional structure is the objective of many researchers. Knowing its structure implies, therefore, to delve into its mechanism of action, information that can be useful in many fields such as biomedicine. 

Crystallography and X -ray diffraction as a tool to solve the structure of proteins is currently the most widespread in the molecular biology of proteins due to the results it provides to the PDB. It is very useful, however, when we talk about membrane proteins we find that there are very few resolved structures. This is because to achieve the crystals we find a series of problems and limitations that we must address and that further complicate the process, therefore it is one of the biggest challenges of current crystallography. 

Developing

As we have explained above, membrane proteins are embedded in the plasma membrane, it is the means that gives them stability.

This implies that in its structure we can distinguish a hydrophobic part, so we must look for alternatives to the solutions used in the crystallization of hydrosoluble proteins. The purification of membrane proteins is a key and critical point of the process, since obtain. 

First approaches

Crambina: It is a small membrane protein, about 46 amino acids, and great stability due to the presence of disulfide bridges. It was first isolated from the seeds of a brassicaceae, Hispanic Subesp rambe. Abyssinica (6). Its structure was resolved by Wayne to. Hendrickson and Martha M. Teer in 1981 using the simple anomalous dispersion method (7). It is a model protein for membrane protein study. The strategy that was used for crystallization was the use of an organic solvent, ethanol. However, it is a strategy that does not work for a large majority of membrane proteins due to cannot be stable in organic solvents (4). 

Hemaglutinine: It is a membrane protein found in the flu virus that allows it to locate and infect host cells (8). Is clearly differentiated by 

Two different parts, the tail, which is located inside the membrane and a globular head, which contain the different antigens and receptors necessary for their mechanism of action. Its structure was resolved in 1981 by Wilson et al. To crystallize this protein, a proteolysis of the Hidrosoluble region was carried out. The inconvenience of this method is that it cannot be crystallized and therefore solve the structure of the transmembrane region. 

Insulin receiver: it is a membrane protein that has a fundamental role in the metabolism of living beings, since it allows insulin to activate or inhibit different signaling routes inside the cell. It is a membrane protein of the tyrosine-kinase type that is characterized by having a portion inside the plasma membrane and another extracellular membrane. As in the example described above to crystallize this protein a proteolysis of the Hidrosoluble region is carried out, which is from which the structure is resolved. In this protein, the use of genetic engineering was also incorporated to eliminate this first phase of proteolysis through the expression of the hydrosoluble region in host cells. 

Current action protocol

In the first place it is important to select the protein to study and clone within a convenient expression system to amplify. Once a considerable amount to carry out the experiments must be obtained, the protein must. Finally, the necessary variables should be considered to create a strategy that allows the macromolecule crystals to be obtained. 

Role of detergents

The most widespread strategy in membrane protein crystallization is the use of detergents. They are amphipatic molecules formed by a polar head and a hydrophobic stem or tail. Depending on the hydrophilic head we can distinguish ionic, nonionic or zwitterionic detergents. Depending on its structure the detergent will have a certain behavior. They act by imitating cell membranes in which proteins are inserted so that they form aggregates or complexes with mycle -shaped membrane proteins that can be solubilized in a solution to which it is added precipitating agent. 

Types of crystals

Membrane proteins form crystals of three types mainly: 2D crystals, 3D type I and 3D type II, the last two appointed types that interest for the studies of x -ray diffraction of X -ray diffraction. 

• Type I crystals: They are characterized by hydrophobic interactions between protein-detergent-lipid. Crystallize following the in meso method or also called the lipid cubic phase. It consists of the crystallization of membrane proteins inside a bicela that creates a stable medium so that crystals can grow in the three dimensions of space. Bicels are discs that mimic plasma membranes in which we can distinguish lipids and amphipatic molecule (detergent).
•  Type II crystals: We can highlight the presence of interactions between the hydrophilic regions of proteins. Crystallize following the in Surfo method that consists in treating the protein-detergent complicus as a soluble protein. This technique gives rise to the formation of micelles and the choice of detergent is crucial, since it will determine how good it will be the diffraction. 

conclusion

Membrane proteins are one of the great challenges in the field of crystallography due to the large number of inconveniences they present. The increase in variables to take into account as well as the impossibility of dissolving them in the solutions used for hydrosoluble proteins are some of the complications we face when we want to crystallize them. However, the growing interest they present in areas such as pharmacology makes new useful strategies and methods that gradually overcome the barriers that occur thus allowing to know and increase the number of resolved structures.