Principles of structural organization of membrane proteins and methods for its prediction for transmembrane proteins. Membrane processes. Plasma membrane Membrane proteins are amphiphilic compounds

If the main role of lipids in membranes is to stabilize the bilayer, then proteins are responsible for the functional activity of membranes. Some of them provide transport of certain molecules and ions, others are enzymes, others are involved in the binding of the cytoskeleton to the extracellular matrix or serve as receptors for hormones, mediators,

eicosanoids, lipoproteins, nitric oxide (NO). Proteins account for 30 to 70% of the mass of membranes. Proteins determine the functioning of each membrane.

Structural features

and localization of proteins in membranes

Membrane proteins in contact with the hydrophobic portion of the lipid bilayer must be amphiphilic. Those parts of the protein that interact with the hydrocarbon chains of fatty acids contain predominantly non-polar amino acids. The protein regions located in the region of the polar “heads” are enriched with hydrophilic amino acid residues.

Localization of proteins in membranes. Transmembrane proteins, for example: 1 - glycophorin A; 2 - adrenaline receptor. Surface proteins: 3 - proteins associated with integral proteins, for example, the enzyme succinate dehydrogenase; 4 - proteins attached to the polar “heads” of the lipid layer, for example, protein kinase C; 5 - proteins “anchored” in the membrane using a short hydrophobic terminal domain, for example, cytochrome b 5; 6 - “anchored” proteins covalently connected to the membrane pipid (for example, the enzyme alkaline phosphatase).

Surface proteins

Surface proteins often attach to the membrane, interacting with integral

proteins or surface areas of the lipid layer.

Proteins that form complexes with integral membrane proteins

A number of digestive enzymes involved in the hydrolysis of starch and proteins are attached to integral membrane proteins of intestinal microvilli.

Examples of such complexes are sucrase-isomaltase and maltase-glycoamylase.

Proteins associated with polar heads of membrane lipids

Polar or charged domains of a protein molecule can interact with the polar heads of lipids, forming ionic and hydrogen bonds. In addition, many proteins soluble in the cytosol can, under certain conditions, bind to the membrane surface for a short time. Sometimes protein binding is a necessary condition for the manifestation of enzymatic activity. Such proteins, for example, include protein kinase C and blood clotting factors.

Fastening with a membrane “anchor”

The “anchor” can be a nonpolar protein domain built from amino acids with hydro-

phobic radicals. An example of such a protein is cytochrome b 5 of the ER membrane. This protein is involved in redox reactions as an electron carrier.

The role of a membrane “anchor” can also be performed by a fatty acid residue covalently bound to the protein (myristic - C 14 or palmitic - C 16). Proteins associated with fatty acids are located mainly on the inner surface of the plasma membrane. Myristic acid adds to the N-terminal glycine to form an amide bond. Palmitic acid forms a thioester bond with cysteine ​​or an ester bond with serine and threonine residues.

A small group of proteins can interact with the outer surface of the cell using a phosphatidylinositol glycan protein covalently attached to the C-terminus of the protein. This “anchor” is often the only link between the protein and the membrane, therefore, under the action of phospholipase C, this protein is separated from the membrane.

Transmembrane (integral) proteins

Some of the transmembrane proteins span the membrane once (glycophorin), others have several regions (domains) that sequentially cross the bilayer.

Transmembrane domains spanning the bilayer have an α-helical conformation. Polar amino acid residues face the inside of the globule, and nonpolar ones are in contact with membrane lipids. Such proteins are called “inverted” in comparison with water-soluble proteins, in which most of the hydrophobic amino acid residues are hidden inside, and the hydrophilic ones are located on the surface.

The charged amino acid radicals within these domains are chargeless and protonated (-COOH) or deprotonated (-NH2).

Glycosylated proteins

Surface proteins or domains of integral proteins, located on the outer surface of all membranes, are almost always glycosylated. Oligosaccharide residues can be attached through the amide group of asparagine or the hydroxyl groups of serine and threonine.

Oligosaccharide residues protect the protein from proteolysis and are involved in ligand recognition or adhesion.

Lateral diffusion of proteins

Some membrane proteins move along the bilayer (lateral diffusion) or rotate around an axis perpendicular to its surface.

The lateral diffusion of integral proteins in the membrane is limited, this is due to their large size, interaction with other membrane proteins, elements of the cytoskeleton or extracellular matrix.

Membrane proteins do not move from one side of the membrane to the other (“flip-flop” jumps), like phospholipids.

Proteins associated with the polar heads of membrane lipids

Proteins that form complexes with integral membrane proteins

Surface proteins

Surface proteins often attach to the membrane, interacting with integral proteins or surface regions of the lipid layer.

A number of digestive enzymes involved in the hydrolysis of starch and proteins are attached to integral proteins of the membranes of intestinal microvilli.

Examples of such complexes are sucrase-isomaltase and maltase-glycoamylase. Perhaps the connection of these digestive enzymes with the membrane allows the hydrolysis of substrates at a high rate and the absorption of hydrolysis products by the cell.

Polar or charged domains of a protein molecule can interact with the polar heads of lipids, forming ionic and hydrogen bonds. In addition, many proteins soluble in the cytosol can, under certain conditions, bind to the membrane surface for a short time. Sometimes protein binding is a necessary condition for the manifestation of enzymatic activity. Such proteins, for example, include protein kinase C and blood clotting factors.

Fastening with a membrane "anchor"

The “anchor” can be a nonpolar protein domain built from amino acids with hydrophobic radicals. An example of such a protein is cytochrome b 5 of the ER membrane. This protein is involved in redox reactions as an electron carrier.

The role of a membrane “anchor” can also be performed by a fatty acid residue covalently bound to the protein (myristic - C 14 or palmitic - C 16). Proteins associated with fatty acids are located mainly on the inner surface of the plasma membrane. Myristic acid adds to the N-terminal glycine to form an amide bond. Palmitic acid forms a thioester bond with cysteine ​​or an ester bond with serine and threonine residues.

A small group of proteins can interact with the outer surface of the cell using a phosphatidylinositol glycan protein covalently attached to the C-terminus of the protein. This “anchor” is often the only link between the protein and the membrane, therefore, under the action of phospholipase C, this protein is separated from the membrane.

Some of the transmembrane proteins span the membrane once (glycophorin), others have several regions (domains) that sequentially cross the bilayer.

Integral membrane proteins containing from 1 to 12 transmembrane domains. 1- LDL receptor; 2 - GLUT-1 - glucose transporter; 3 - insulin receptor; 4 - adrenoreceptor.

Transmembrane domains spanning the bilayer have an α-helical conformation. Polar amino acid residues face the inside of the globule, and nonpolar ones are in contact with membrane lipids. Such proteins are called “inverted” in comparison with water-soluble proteins, in which most of the hydrophobic amino acid residues are hidden inside, and the hydrophilic ones are located on the surface.

Lipids in membranes are primarily responsible for their structural properties - they create a bilayer, or matrix, in which the active components of the membrane - proteins - are located. It is proteins that give various membranes their uniqueness and provide specific properties. Numerous membrane proteins perform the following main functions: they determine the transfer of substances across membranes (transport functions), carry out catalysis, provide the processes of photo- and oxidative phosphorylation, DNA replication, translation and modification of proteins, signal reception and transmission of nerve impulses, etc.

It is customary to divide membrane proteins into 2 groups: integral(internal) and peripheral(external). The criterion for such separation is the degree of strength of binding of the protein to the membrane and, accordingly, the degree of severity of processing necessary to extract the protein from the membrane. Thus, peripheral proteins can be released into solution even when membranes are washed with buffer mixtures with low ionic strength, low pH values ​​in the presence of chelating substances, such as ethylenediaminetetraacetate (EDTA), which bind divalent cations. Peripheral proteins are released from membranes under such mild conditions because they are associated with lipid heads or with other membrane proteins using weak electrostatic interactions, or with hydrophobic interactions with lipid tails. On the contrary, integral proteins are amphiphilic molecules, have large hydrophobic regions on their surface and are located inside the membrane, so their extraction requires destruction of the bilayer. For these purposes, detergents or organic solvents are most often used. The methods for attaching proteins to the membrane are quite varied (Fig. 4.8).

Transport proteins. The lipid bilayer is an impermeable barrier to most water-soluble molecules and ions, and their transport across biomembranes depends on the activity of transport proteins. There are two main types of these proteins: channels(pores) and carriers. Channels are membrane-crossing tunnels in which binding sites for transported substances are accessible on both membrane surfaces simultaneously. Channels do not undergo any conformational changes during the transport of substances; their conformation changes only when opening and closing. Carriers, on the contrary, change their conformation during the transfer of substances across the membrane. Moreover, at any given time, the binding site of the transported substance in the carrier is accessible only on one surface of the membrane.

Channels, in turn, can be divided into two main groups: voltage-dependent and chemically regulated. An example of a potential-dependent channel is the Na + channel; its operation is regulated by changing the electric field voltage. In other words, these channels open and close in response to change transmembrane potential. Chemically regulated channels

open and close in response to the binding of specific chemical agents. For example, the nicotinic acetylcholine receptor, when a neurotransmitter binds to it, goes into an open conformation and allows monovalent cations to pass through (subsection 4.7 of this chapter). The terms “pore” and “channel” are usually used interchangeably, but pores are more often understood as non-selective structures that distinguish substances mainly by size and allow passage of all sufficiently small molecules. Channels are often understood as ion channels. The transport rate through the open channel reaches 10 6 - 10 8 ions per second.

Transporters can also be divided into 2 groups: passive and active. With the help of passive carriers, one type of substance is transported across the membrane. Passive transporters are involved in facilitated diffusion and only increase the flow of substances along an electrochemical gradient (for example, the transfer of glucose across erythrocyte membranes). Active carriers transport substances across the membrane using energy. These transport proteins accumulate substances on one side of the membrane, transporting them against the electrochemical gradient. The speed of transport using carriers depends very much on their type and ranges from 30 to 10 5 s -1. The terms “permease” and “translocase” are often used to designate individual transporters, which can be considered synonymous with the term “transporter”.

Enzyme functions of membrane proteins. A large number of different enzymes function in cell membranes. Some of them are localized in the membrane, finding there a suitable environment for the transformation of hydrophobic compounds, others, thanks to the participation of membranes, are located in them in strict order, catalyzing successive stages of vital processes, while others require the assistance of lipids to stabilize their conformation and maintain activity. Enzymes were found in biomembranes - representatives of all known classes. They can penetrate the membrane through, be present in it in dissolved form, or, being peripheral proteins, bind to membrane surfaces in response to any signal. The following characteristic types of membrane enzymes can be distinguished:

1) transmembrane enzymes that catalyze coupled reactions on opposite sides of the membrane. These enzymes usually have several active centers located on opposite sides of the membrane. Typical representatives of such enzymes are components of the respiratory chain or photosynthetic redox centers that catalyze redox processes associated with electron transport and the creation of ion gradients on the membrane;

2) transmembrane enzymes involved in the transport of substances. Transport proteins that couple substance transfer with ATP hydrolysis, for example, have a catalytic function;

3) enzymes that catalyze the transformation of membrane-bound substrates. These enzymes are involved in the metabolism of membrane components: phospholipids, glycolipids, steroids, etc.

4) enzymes involved in the transformation of water-soluble substrates. With the help of membranes, most often in an attached state, enzymes can concentrate in those areas of the membrane where the content of their substrates is greatest. For example, enzymes that hydrolyze proteins and starch are attached to the membranes of intestinal microvilli, which helps to increase the rate of breakdown of these substrates.

Cytoskeletal proteins . The cytoskeleton is a complex network of protein fibers of various types and is present only in eukaryotic cells. The cytoskeleton provides mechanical support for the plasma membrane and can determine the shape of the cell, as well as the location of organelles and their movement during mitosis. With the participation of the cytoskeleton, such important processes for the cell as endo- and exocytosis, phagocytosis, and amoeboid movement are also carried out. Thus, the cytoskeleton is the dynamic framework of the cell and determines its mechanics.

The cytoskeleton is formed from three types of fibers:

1) microfilaments(diameter ~6 nm). They are thread-like organelles - polymers of the globular protein actin and other proteins associated with it;

2) intermediate filaments (diameter 8-10 nm). Formed by keratins and related proteins;

3) microtubules(diameter ~ 23 nm) - long tubular structures.

They consist of a globular protein called tubulin, the subunits of which form a hollow cylinder. The length of microtubules can reach several micrometers in the cytoplasm of cells and several millimeters in the axons of nerves.

The listed cytoskeletal structures penetrate the cell in different directions and are closely associated with the membrane, attaching to it at some points. These sections of the membrane play an important role in intercellular contacts; with their help, cells can attach to the substrate. They also play an important role in the transmembrane distribution of lipids and proteins in membranes.

The formation of transmembrane proteins must include the stages of recognition of transmembrane domains and their integration into the lipid bilayer

Transmembrane domains extend laterally from the translocon through the protein-lipid interface

Positioning on the translocation channel and start transfer of secretory and transmembrane proteins happen the same way. However, the translocation of membrane proteins must be combined with their integration or insertion into the ER lipid bilayer. Integration occurs at the moment when the transmembrane domains are recognized by the translocon, then their translocation into the ER lumen stops, and they begin to be transferred from the channel into the lipid bilayer in the lateral direction. Many different types of transmembrane proteins are synthesized and integrated in this way, including those that span the membrane several times.

The first step on the protein integration pathway into the membrane is the recognition of transmembrane domains by the translocon. These domains extend over a distance of about twenty hydrophobic amino acids. Due to their hydrophobic nature, some transmembrane domains are recognized by SRP as signal sequences. These so-called signal anchor sequences first position the nascent protein on the ER and are then directed into the channel like normal signal sequences.

However signal anchor sequences are not cleaved from the protein, but are integrated into the membrane. As shown in the figure below, unlike the signal anchor sequence, most transmembrane domains are recognized by the translocon as soon as they leave the ribosome, after completion of targeting by a conventional N-terminal signal sequence. Information that the transmembrane domain has already been synthesized must be transferred to the translocon by a route other than transfer from SRP.

The simplest sign, indicating that the transmembrane domain is located in the translocon is the hydrophobicity of the domain itself. Due to the peculiarity of its structure, the translocation channel exhibits this hydrophobicity. As shown in Fig. 3.21, the structure of the translocon suggests that the channel is capable of opening like a clam shell, allowing the transmembrane domain to simultaneously contact the channel and the lipid bilayer. Apparently, signal sequences and transmembrane domains bind to the Sec61a protein located on the side of the opening valves, and this binding then causes the lateral opening of the channel.

This scheme has been proposed based on experimental data, according to which the transmembrane domains in the channel contact Sec61a and . As a result, although the translocon contains a water channel, there are enough hydrophobic channels in the membrane in which translocated polypeptides can enter the lipid environment. It is expected that regions containing polar amino acids should move through the channel without stopping, while hydrophobic domains, due to strong interactions with lipids, will remain associated with the side walls of the channel, preventing translocation.

In some cases translocon may identify the transmembrane domain differently. For example, sometimes during the synthesis of a transmembrane domain, changes in the interaction between the ribosome and the translocon occur before the domain leaves the ribosome. These changes serve as a signal to the translocon about the imminent appearance of a transmembrane domain. How the transmembrane domain causes changes in the ribosome and transfers them to the translocon remains unclear. Sometimes polar elements of the newly formed chain adjacent to the transmembrane domain are also required for recognition. This suggests that, at least in some cases, the recognition process must involve more than just a hydrophobic interaction between the domain and the lipid channel environment.

Boundary of the channel and the lipid layer, apparently, serves as a pathway for the exit of transmembrane domains from the channel after their recognition. However, the mechanism for domain exit from the translocon varies somewhat from substrate to substrate. Some domains leave the translocon almost immediately after being recognized in the channel. In these cases, the transmembrane domain first contacts Sec61a and lipids, and then only lipids; it is assumed that the domain has already penetrated into the lipid bilayer.

For integration of such domains of other proteins, with the exception of the Sec61 complex, is not required. Other transmembrane domains integrate more slowly and, after recognition, do not leave the translocon for a long time, sometimes even remaining there until the end of translation. As they exit the channel into the bilayer, these transmembrane domains come into contact with the TRAM protein, although its further role remains unclear.

Degree of hydrophobicity partly determines whether the transmembrane domain is integrated immediately or occurs at a later stage in protein synthesis. More hydrophobic domains can move more quickly into the lipid bilayer, but less hydrophobic ones can remain at the interface and require additional transport factors. It is possible that TRAM and other proteins serve as chaperones for some transmembrane domains. They promote the integration of domains whose hydrophobicity is insufficient for movement.

It's obvious that at least group of transmembrane proteins may be multiple forms containing a specific domain, which in some cases is integrated into the membrane, and in others remains unrecognized. Proteins such as TRAM may determine under what conditions such substitutions will be integrated.