Solving the structure of membrane proteins

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Solving the structure of membrane proteins
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bet | 9/13 | Sana | 16.05.2024 | Hajmi | 1,48 Mb. | | #237391 |
Bog'liq Structure and function of biological membranes.2.3. Solving the structure of membrane proteins.
In order to understand more fully the mechanisms of action of membrane proteins such as the transporters described here, we can determine their three-dimensional protein structures. As a result of huge advances in structural biology in the last 50 years, we now have access to many thousands of protein structures in online databases. This enables researchers to visualize the structure of their protein of interest, and thus gain insight into its mechanism.
X-ray crystallography The structure of whale myoglobin was solved in 1958 using X-ray crystallography, earning John C. Kendrew and Max Perutz the Nobel Prize in Chemistry. This was the first protein structure to be solved using this technique, and since then thousands of proteins have been solved using this method. X-ray crystallography works by firing a beam of X-rays at a crystalline structure and measuring the diffraction of the X-rays after they have passed through the structure of interest. This generates an electron density map, showing where different atoms in the structure are located. For regular crystalline solids such as salts this is relatively straightforward, but for large irregular molecules such as proteins it can present many technical challenges. Before a protein is subjected to X-ray beams, it must first be purified and crystallized. In nature, proteins exist in the busy milieu of a cell, surrounded by thousands of other types of proteins, as well as lipids and other molecules. A common method of obtaining enough of the protein of interest involves expressing the relevant gene in a system such as bacteria. The gene is tagged with a small protein tag which can be used to isolate the protein of interest. Bacterial systems allow large amounts of protein to be produced cheaply and quickly. However, if the protein of interest is from a species that is only distantly related to that in which it is normally expressed (e.g. a human protein produced in Escherichia coli (E. coli)), the lack of correct glycosylation enzymes and the differences in protein folding and assembly may prevent the production of a biologically active protein. In addition, the expression of membrane proteins that make pores or channels can kill the host organism.
A pure protein sample is then crystallized by allowing water to evaporate away, in exactly the same way as a solution of salt will form crystals naturally when left to dry. Optimum conditions for this must be determined, and crystallization conditions are not always straightforward, as they differ from one protein to another. For soluble proteins such as myoglobin this is easier than for insoluble membrane proteins. Membrane proteins have lipid-soluble domains that will not dissolve in an aqueous medium. This significantly decreases the ease with which membrane protein structures can be solved using X-ray diffraction. However, there are ways in which scientists can overcome this difficulty. Generally, membrane proteins are removed from the membrane in which they were made and placed in an environment of lipids and detergents for crystallization. Sometimes the lipids associated with the protein are apparent in the crystal structure.
The number of solved crystal structures of proteins is constantly growing as technology improves and expertise is shared among scientists to help to optimize conditions for crystal production. The Protein Data Bank (PDB) is an online archive of protein structures which can be freely accessed by scientists worldwide. At the time of writing, 88% of the structures in the PDB have been solved by X-ray crystallography, and there are currently just under 70 000 X-ray crystal structures in the database. The number of membrane protein structures in the PDB is increasing rapidly with the refinement of crystallization techniques .
Other structural techniques Nuclear magnetic resonance (NMR) spectroscopy is another valuable technique for elucidating membrane protein structure. Molecules are placed in a magnetic field and the resonance properties of different atomic nuclei are measured, which gives an indication of where in a particular molecule different atoms are located. Generally, NMR is limited to smaller proteins, below around 35 kDa in size. It also offers the potential to visualize proteins in a more physiologically relevant environment (e.g. in lipid bilayers or micelles). Another advantage of NMR is that it does not require the protein to be locked in a crystal lattice—a structure which can distort the natural shape of the protein.
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