[towersimper: This article is a translation, only for research, not for commercial development, please indicate the translator towersimper and the original author Robert Michael Stroud]
Membrane proteins account for almost 40% of all known proteins, including receptors, channel proteins, and signaling molecules, which are necessary for cell communication. If the membrane protein fails, it will cause many diseases. However, to date, membrane proteins account for only a small portion of the total known protein structure. Structure-based design is a powerful weapon for drug development, and for the drug to be effectively treated, the site of action must be precise and side effects minimized. X-ray crystallography is still the only general method for solving the atomic structure of proteins of different sizes. However, since it is very difficult to prepare high-purity membrane proteins and obtain their crystals, this method cannot be implemented.
This is a practical problem because hydrophilic proteins, such as those located in the cytoplasm, crystallize relatively easily in solution, but membrane proteins contain hydrophobic regions located in the lipid layer. In order to maintain their shape, these lipophilic domains must be surrounded by components similar to those found on natural cell membranes, which makes it difficult to obtain crystals with good diffraction properties. However, in the past two years, some technological advancements have enabled people to determine the structure of membrane proteins.
Extraction and stabilization
Technological advancements are the result of improvements to multiple steps in the crystallization process. An example comes from Raymond Stevens of the Scripps Research Institute and his colleagues. They found that lipids determine the structure of G protein-coupled receptors (GPCRs) that respond to adrenaline. Is required. When Stevens tried to crystallize GPCRs, he found that cholesterol molecules are necessary for crystal formation, and from the crystal structure, cholesterol acts as an adhesive between GPCR dimer molecules [1]. This can also be explained structurally from the following observations: On the cell membrane, cholesterol is necessary for the receptor GPCR to form dimers and perform signal transduction functions.
The use of detergents to separate membrane proteins has a profound effect on their ability to form crystals. Typical detergents, such as
Beta-octyl glutamic acid (beta-octyl glucoside), which can produce larger lipid globules (also known as micelles), contains a single layer of phospholipids and a single phospholipid tail Facing inward. Large micelles increase the ratio of lipid to protein, making it difficult for membrane proteins to aggregate to a density suitable for crystal formation. Stevens worked with some chemists to design new detergents, such as amphoteric molecules based on cholate, which can produce smaller micelles, so that the membrane protein molecules are more tightly aggregated, resulting in better crystals [2].
The third factor that improves crystal formation is the origin of the protein. The quantity and quality of protein production depends on the organism, cell type, promoter and carrier used to express the protein. However, it is not yet possible to know in advance which biological species or which expression method can produce better performing proteins. Since many membrane proteins are expressed in multiple biological species, homologous genes should be tested and screened to find the most suitable protein for crystal formation [3]. Researchers can now use high-throughput methods to screen out the best conditions among a large number of protein expression and purification conditions, which can help speed up the process of finding suitable and crystal-forming experiments [4].
A notable example of this optimization method comes from the Rod MacKinnon laboratory at Rockefeller University. Because of their interest in the mechanism of the CLC chloride transporter, the researchers at the laboratory improved the preparations made from them by replacing each amino acid present in natural conditions with a methionine at each of the 30 sites of the protein. The data obtained in the crystal. They then labeled the heavy element selenium with these methionines to help verify the atomic structure of the protein more accurately.
Redesigned protein
Chopping off the ends of proteins is a trick that people often use to make proteins in the cytoplasm easier to form crystals. Similarly, removing the hydrophilic regions and flexible ends of membrane proteins also promotes crystal formation. Human aquaporin 4, HA4 is associated with the autoimmune disease neuromyelitis optica, which is similar to amyotrophic lateral sclerosis (ALS). By cutting out the flexible area of ​​HA4, we obtained a high-definition crystal structure [5]. Another method is to introduce mutations, change the protein sequence to stabilize a specific protein conformation, and after screening to find mutations that may fix other flexible regions.
The use of more drastic design methods—large-scale gene redesign (wholesale gene redesign) —makes it possible to analyze the structure of proteins that cannot be studied by other methods. Some amino acids have multiple codons—nucleotide triplets make up the genetic code—and different biological species have a preference for different codons that produce the same protein. Because codon preference affects protein translation efficiency, taking a gene from one organism and expressing it in another organism may improve protein production. We recently designed a gene for the aquaporin of Plasmodium falciparum and expressed it in the plasma membrane component of Escherichia coli. We change the gene sequence of Plasmodium falciparum by using E. coli-preferred codons. This process is also called codon optimization. Despite changes in the DNA sequence, the protein sequence expressed remains unchanged [6].
The rapid development of technology in recent years has made the prospects for determining the structure of membrane proteins and how they function more bright. We can now begin to explore the mechanisms of transmembrane processes that are associated with various diseases, such as cancer, diabetes, schizophrenia, and depression. In these diseases, the signal transduction function of membrane proteins It is very difficult to study defects at the atomic structure level. \ Life Science Forum
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