Every cell in our body is a protein production factory. Some of them are produced for internal use, to support the life of the cell, and the other part is “exported”. All properties of protein molecules (including the ability to amazingly precisely catalyze the transformations of other molecules in the cell) depend on the spatial structure of the protein, and the structure of each protein is unique.
The spatial structure is formed by a unique arrangement of the protein chain, consisting of different amino acid residues (beads of different colors - Fig. 1). The sequence of amino acids in a protein chain is determined by its genome and synthesized by the ribosome, after which the spatial structure of the chain is formed “by itself” during the folding of the protein chain, which leaves the ribosome still practically disordered.
The formation of a unique protein globule from a disordered chain (as well as its unfolding) requires overcoming a “barrier” that has the form of an unstable “half-folded” globule (Fig. 1)
Alexey FinkelshteinThis chain is folded by the interaction of its amino acids, and into the same structure - both in the body and in a test tube. The variety of possible layouts of the same chain is unimaginably large. But a given amino acid sequence usually has only one stable (“correct”) structure, which gives the protein its unique properties. It is stable because it is the one that has the minimum energy.
The same principle operates in the formation of crystals: the substance acquires the structure in which the bond energy is minimal.
What do proteins and the Universe have in common?
Here, the scientists faced a question: how can a protein chain spontaneously “find” its only stable structure, if searching through the colossal number of all options (about 10,100 for a chain of 100 amino acid residues) would take more time than the lifetime of the Universe. This “Levinthal paradox”, formulated half a century ago, has only now been resolved. To solve it, it was necessary to use methods of theoretical physics.
Crystals of various proteins grown on the Mir space station and during NASA shuttle flights
NASA Marshall Space Flight CenterScientists from the Institute of Protein of the Russian Academy of Sciences (IB) have created a theory of the rates of formation of spatial structures of protein molecules. The results of the work were recently published in journals Atlas of Science , Chem Phys Chem And "Biophysics". Job supported a grant from the Russian Science Foundation (RSF).
“The ability of proteins to spontaneously form their spatial structures in a matter of seconds or minutes is a long-standing mystery of molecular biology.
Our work presents a physical theory that allows us to estimate the speed of this process depending on the size of proteins and the complexity of their structure,” begins the story about his work, Corresponding Member of the Russian Academy of Sciences, Doctor of Physical and Mathematical Sciences, Chief Researcher of the Protein Institute of the Russian Academy of Sciences, head of the RSF grant Alexey Finkelstein.
“It has long been known that a protein chain acquires its unique structure under certain environmental conditions, and under others (for example, when a solution is acidified or heated), this structure unfolds. At the intersection of these conditions, the unique structure of the protein is in dynamic equilibrium with the unfolded shape of its chain, he continues. “The processes of folding and unfolding coexist there, and their physics is most transparent. Therefore, we focused precisely on such equilibrium and quasi-equilibrium conditions - in contrast to other researchers who seemed to reasonably (but erroneously, as it turned out) believe that the path to the secret of protein folding should be sought where it occurs most quickly.”
Unwrapping the protein is a good start, but not the answer.
“The first approach to Levinthal’s problem was developed by us a long time ago,” says Alexey Finkelstein, “and was as follows: since it is very difficult to theoretically trace the path of protein folding, we need to study the process of its unfolding. It sounds paradoxical, but in physics there is a principle of “detailed equilibrium”, which states: any process in an equilibrium system proceeds along the same path and at the same speed as its opposite. And since in dynamic equilibrium the rates of folding and unfolding are the same, we examined a simpler process of protein unfolding (after all, breaking it is easier than making it) and characterized that “barrier” (see picture 1), the instability of which determines the rate of the process.”
Following the principle of detailed equilibrium, scientists from the Institute of Protein of the Russian Academy of Sciences assessed both “from above” and “from below” the rate of folding of proteins - both large and small, both with simple and complex chain packing. Small and simply structured proteins fold faster (speed rating “from above”), while large and/or complexly structured proteins fold more slowly (speed rating “from below”). The values of all other possible folding speeds are contained between them.
However, not all biologists were satisfied with the solution obtained, since, firstly, they were interested in the path of folding (and not unfolding) of the protein, and secondly, the physical “principle of detailed equilibrium” was apparently poorly understood by them.
And the work continued: this time, scientists from the Institute of Biology of the Russian Academy of Sciences calculated the complexity of protein folding. It has long been known that interactions in proteins are associated mainly with so-called secondary structures. Secondary structures are standard, fairly large local "building blocks" of protein structure, determined mainly by the local amino acid sequences within them. The number of possible options for arranging such blocks in the structure of a folded protein can be calculated, which was done by scientists from the Institute of Biochemistry of the Russian Academy of Sciences. The number of such variants is huge - about 10 10 (but far from 10 100!) for a chain of about 100 amino acids, and a protein chain can, according to theoretical estimates, “scan” them in minutes or, for longer chains, in hours. This was how the highest estimate of protein folding time was obtained.
Regular secondary structure - alpha helix
WillowWThe results obtained by two methods (i.e., by analyzing both unfolding and folding of the protein) converge and confirm each other.
“Our work is of fundamental importance for the design of new proteins in the future for the needs of pharmacology, bioengineering, and nanotechnology,” concludes Alexey Finkelstein.
“Questions about the rate of protein folding are relevant when it comes to predicting the structure of a protein from its amino acid sequence, and especially when it comes to the design of new proteins that do not occur in nature.”
“What changed after receiving the RSF grant? The opportunity has arisen to purchase new modern equipment and reagents for work (after all, our laboratory is mainly experimental, although I only spoke here about our theoretical work). But the main thing is that the RSF grant allowed specialists to engage in science, rather than look for part-time work on the side or in distant lands,” says Alexey Finkelshtein.
folding, etc. "protein folding- The process of folding a polypeptide chain into the correct spatial structure. Individual proteins, products of the same gene, have an identical amino acid sequence and acquire the same conformation and function under the same cellular conditions. For many proteins that have a complex spatial structure, folding occurs with the participation "chaperones"
Reactivation of ribonuclease. the process of protein denaturation can be reversible. This discovery was made while studying the denaturation of ribonuclease, which cleaves bonds between nucleotides in RNA. Ribonuclease is a globular protein containing one polypeptide chain consisting of 124 amino acid residues. Its conformation is stabilized by 4 disulfide bonds and many weak bonds.
Treatment of ribonuclease with mercaptoethanol leads to the cleavage of disulfide bonds and the reduction of SH groups of cysteine residues, which disrupts the compact structure of the protein. The addition of urea or guanidine chloride leads to the formation of randomly folded polypeptide chains devoid of ribonuclease. denaturation of the enzyme. If ribonuclease is purified from denaturing agents and mercaptoethanol by dialysis, the enzymatic activity of the protein is gradually restored. This process is called renaturation
The possibility of reactivation has been proven for other proteins. a necessary condition for restoring its conformation is the integrity of the primary structure of the protein.
proteins capable of binding to proteins that are in an unstable, aggregation-prone state, capable of stabilizing their conformation, ensuring protein folding, are called "chaperones".
The role of chaperones in protein folding
During the period of protein synthesis on the ribosome, the protection of reactive radicals is carried out by Sh-70. The folding of many high-molecular proteins with a complex conformation is carried out in the space formed by Sh-60. Sh-60 functions as an oligomeric complex consisting of 14 subunits. The chaperone complex has a high affinity for proteins, on the surface of which there are areas enriched with hydrophobic radicals). Once in the cavity of the chaperone complex, the protein binds to hydrophobic radicals of the apical sections of Sh-60.
The role of chaperones in protecting cell proteins from denaturing stress influences
Chaperones involved in the protection of cellular proteins from denaturing influences are classified as heat shock proteins. Under action (high temperature, hypoxia, infection, ultraviolet radiation, change in pH of the environment, change in the molarity of the environment, the effect of toxic chemicals, heavy metals) the synthesis of HSPs in cells increases . they can prevent their complete denaturation and restore the native conformation of proteins.
Diseases associated with violation
protein folding Alzheimer's disease- amyloidosis of the nervous system, affecting the elderly and characterized by progressive memory impairment and complete personality degradation. Amyloid, a protein that forms insoluble fibrils, disrupts the structure and function of nerve cells, is deposited in brain tissue.
Prion proteins a special class of proteins with infectious properties. Once in the human body, they can cause severe incurable diseases of the central nervous system, called prion diseases. The prion protein is encoded by the same gene as its normal counterpart, i.e. they have identical primary structure. However, the two proteins have different conformations: the prion protein is characterized by a high content of β-sheets, while the normal protein has many helical regions. prion protein is resistant to proteases.
During the synthesis of polypeptide chains, their transport through membranes, and during the assembly of oligomeric proteins, intermediate unstable conformations that are prone to aggregation arise. The newly synthesized polypeptide has many hydrophobic radicals, which are hidden inside the molecule in a three-dimensional structure. Therefore, during the formation of the native conformation, reactive amino acid residues of some proteins must be separated from the same groups of other proteins.
In all known organisms, from prokaryotes to higher eukaryotes, proteins have been found that can bind to proteins that are in an unstable state prone to aggregation. They are able to stabilize their conformation, ensuring protein folding. These proteins are called "chaperones".
1. Classifications of chaperones (III)
According to molecular weight, all chaperones can be divided into 6 main groups:
high molecular weight, with a molecular weight from 100 to 110 kDa;
Sh-90 - with a molecular weight from 83 to 90 kDa;
Sh-70 - with a molecular weight from 66 to 78 kDa;
low molecular weight chaperones with a molecular weight from 15 to 30 kDa.
Among the chaperones there are distinguished: constitutive proteins (the high basal synthesis of which does not depend on stress effects on the cells of the body), and inducible proteins, the synthesis of which is weak under normal conditions, but increases sharply under stress effects on the cell. Inducible chaperones are classified as “heat shock proteins”, the rapid synthesis of which is observed in almost all cells that are exposed to any stress. The name "heat shock proteins" arose from the fact that these proteins were first discovered in cells that were exposed to high temperatures.
2. The role of chaperones in protein folding
During protein synthesis, the N-terminal region of the polypeptide is synthesized earlier than the C-terminal region. To form the conformation of a protein, its complete amino acid sequence is required. Therefore, during protein synthesis on the ribosome, protection of reactive radicals (especially hydrophobic ones) is carried out by Sh-70.
Sh-70 is a highly conserved class of proteins that is present in all parts of the cell: cytoplasm, nucleus, ER, mitochondria. In the region of the carboxyl end of the single polypeptide chain of chaperones there is a region formed by amino acid radicals in the form of a groove. It is capable of interacting with sections of protein molecules and unfolded polypeptide chains 7-9 amino acids long, enriched with hydrophobic radicals. In the synthesized polypeptide chain, such regions occur approximately every 16 amino acids.
Folding of many high-molecular proteins with a complex conformation (for example, domain structure) occurs in a special space formed by Sh-60. Ш-60 function as an oligomeric complex consisting of 14 subunits (Fig. 1-23).
Ш-60 form 2 rings, each of which consists of 7 subunits connected to each other. The Ш-60 subunit consists of 3 domains: apical (apical), intermediate and equatorial. The apical domain has a number of hydrophobic residues facing the cavity of the ring formed by the subunits. The equatorial domain has an ATP binding site and has ATPase activity, i.e. capable of hydrolyzing ATP to ADP and H 3 PO 4.
The chaperone complex has a high affinity for proteins, on the surface of which there are elements characteristic of unfolded molecules (primarily areas enriched in hydrophobic radicals). Once in the cavity of the chaperone complex, the protein binds to hydrophobic radicals of the apical sections of Sh-60. In the specific environment of this cavity, in isolation from other molecules of the cell, possible protein conformations are searched until a single, energetically most favorable conformation is found.
The release of the protein with the formed native conformation is accompanied by ATP hydrolysis in the equatorial domain. If the protein has not acquired its native conformation, then it enters into repeated contact with the chaperone complex. This chaperone-dependent protein folding requires a large amount of energy.
Thus, the synthesis and folding of proteins occur with the participation of different groups of chaperones, which prevent unwanted interactions of proteins with other cellular molecules and accompany them until the final formation of the native structure.
4. Diseases associated with protein misfolding
Calculations have shown that only a small part of the theoretically possible variants of polypeptide chains can take on one stable spatial structure. Most of these proteins can take on many conformations with approximately the same Gibbs energy, but with different properties. The primary structure of most known proteins selected by evolution provides exceptional stability to a single conformation.
However, some water-soluble proteins, when conditions change, can acquire the conformation of poorly soluble molecules capable of aggregation, forming fibrillar deposits in cells called amyloid (from the Latin. amylum - starch). Like starch, amyloid deposits are detected by staining tissue with iodine. This may happen:
with overproduction of certain proteins, resulting in an increase in their concentration in the cell;
when proteins enter cells or form in them that can affect the conformation of other protein molecules;
upon activation of proteolysis of normal body proteins, with the formation of insoluble fragments prone to aggregation;
as a result of point mutations in the protein structure.
As a result of amyloid deposition in organs and tissues, the structure and function of cells are disrupted, their degenerative changes and the proliferation of connective tissue or glial cells are observed. Diseases called amyloids develop. Each type of amyloidosis is characterized by a specific type of amyloid. Currently, more than 15 such diseases have been described.
Alzheimer's disease
Alzheimer's disease is the most frequently noted amyloidosis of the nervous system, usually affecting the elderly and characterized by progressive memory impairment and complete personality degradation. β-amyloid, a protein that forms insoluble fibrils, disrupts the structure and function of nerve cells, is deposited in brain tissue. β-amyloid is a product of changes in the conformations of normal proteins in the human body. It is formed from a larger precursor by partial proteolysis and is synthesized in many tissues. α-Amyloid, in contrast to its normal predecessor, which contains many α-helical regions, has a secondary α-folded structure, aggregates to form insoluble fibrils, and is resistant to the action of proteolytic enzymes.
The reasons for the disruption of folding of native proteins in brain tissue remain to be elucidated. It is possible that with age, the synthesis of chaperones capable of participating in the formation and maintenance of native protein conformations decreases, or the activity of proteases increases, which leads to an increase in the concentration of proteins prone to change conformation.
Prion diseases
Prions are a special class of proteins that have infectious properties. When they enter the human body or spontaneously arise in it, they can cause severe incurable diseases of the central nervous system, called prion diseases. The name "prions" comes from the abbreviation of the English phrase proteinaceous infectious particle- protein infectious particle.
The prion protein is encoded by the same protein as its normal counterpart, i.e. they have identical primary structure. However, the two proteins have different conformations: the prion protein is characterized by a high content of α-sheets, while the normal protein has many α-helical regions. In addition, the prion protein is resistant to the action of proteases and, entering the brain tissue or being formed there spontaneously, promotes the conversion of a normal protein into a prion protein as a result of protein-protein interactions. A so-called “polymerization core” is formed, consisting of aggregated prion proteins, to which new normal protein molecules are able to attach. As a result, conformational rearrangements characteristic of prion proteins occur in their spatial structure.
There are known cases of hereditary forms of prion diseases caused by mutations in the structure of this protein. However, it is also possible for a person to become infected with prion proteins, resulting in a disease that leads to the death of the patient. Thus, kuru is a prion disease of the natives of New Guinea, the epidemic nature of which is associated with traditional cannibalism in these tribes and the transfer of infectious protein from one individual to another. Due to changes in their lifestyle, this disease has practically disappeared.
Biological chemistry Lelevich Vladimir Valeryanovich
Folding
Protein folding is the process of folding a polypeptide chain into the correct spatial structure. In this case, distant amino acid residues of the polypeptide chain are brought closer together, leading to the formation of a native structure. This structure has unique biological activity. Therefore, folding is an important stage in the transformation of genetic information into the mechanisms of cell functioning.
Structure and functional role of chaperones in protein folding
During the synthesis of polypeptide chains, their transport through membranes, and during the assembly of oligomeric proteins, intermediate unstable conformations that are prone to aggregation arise. The newly synthesized polypeptide has many hydrophobic radicals, which are hidden inside the molecule in a three-dimensional structure. Therefore, during the formation of the native conformation, reactive amino acid residues of some proteins must be separated from the same groups of other proteins.
In all known organisms, from prokaryotes to higher eukaryotes, proteins have been found that can bind to proteins that are in an unstable state prone to aggregation. They are able to stabilize their conformation, ensuring protein folding. These proteins are called chaperones.
Classification of chaperones (III)
According to molecular weight, all chaperones can be divided into 6 main groups:
1. high molecular weight, with a molecular weight from 100 to 110 kDa;
2. Sh-90 – with a molecular weight from 83 to 90 kDa;
3. Sh-70 – with a molecular weight from 66 to 78 kDa;
6. Low molecular weight chaperones with a molecular weight from 15 to 30 kDa.
Among the chaperones there are distinguished: constitutive proteins (the high basal synthesis of which does not depend on stress effects on the cells of the body), and inducible proteins, the synthesis of which is weak under normal conditions, but increases sharply under stress effects on the cell. Inducible chaperones belong to the “heat shock proteins”, the rapid synthesis of which is observed in almost all cells that are exposed to any stress. The name “heat shock proteins” arose from the fact that these proteins were first discovered in cells that were exposed to high temperatures.
The role of chaperones in protein folding
During protein synthesis, the N-terminal region of the polypeptide is synthesized earlier than the C-terminal region. To form the conformation of a protein, its complete amino acid sequence is required. Therefore, during protein synthesis on the ribosome, protection of reactive radicals (especially hydrophobic ones) is carried out by Sh-70.
Sh-70 is a highly conserved class of proteins that is present in all parts of the cell: cytoplasm, nucleus, mitochondria.
Folding of many high-molecular proteins with a complex conformation (for example, domain structure) occurs in a special space formed by Sh-60. Sh-60 functions as an oligomeric complex consisting of 14 subunits.
The chaperone complex has a high affinity for proteins, on the surface of which there are elements characteristic of unfolded molecules (primarily areas enriched in hydrophobic radicals). Once in the cavity of the chaperone complex, the protein binds to hydrophobic radicals of the apical sections of Sh-60. In the specific environment of this cavity, in isolation from other molecules of the cell, a selection of possible protein conformations occurs until a single, energetically most favorable conformation is found.
The release of the protein with the formed native conformation is accompanied by ATP hydrolysis in the equatorial domain. If the protein has not acquired its native conformation, then it enters into repeated contact with the chaperone complex. This chaperone-dependent protein folding requires more energy.
Thus, the synthesis and folding of proteins occurs with the participation of different groups of chaperones, which prevent unwanted interactions of proteins with other cellular molecules and accompany them until the final formation of the native structure.
The role of chaperones in protecting cell proteins from denaturing stress influences
Chaperones involved in the protection of cellular proteins from denaturing influences, as mentioned above, are classified as heat shock proteins (HSPs) and are often referred to in the literature as HSPs (heat shock proteins).
Under the influence of various stress factors (high temperature, hypoxia, infection, ultraviolet radiation, changes in the pH of the environment, changes in the molarity of the environment, the effect of toxic chemicals, heavy metals, etc.), the synthesis of HSPs in cells increases. Having a high affinity for the hydrophobic regions of partially denatured proteins, they can prevent their complete denaturation and restore the native conformation of proteins.
It has been established that short-term stress increases the production of HSP and increases the body's resistance to long-term stress. Thus, short-term ischemia of the heart muscle during running with moderate training significantly increases the resistance of the myocardium to long-term ischemia. Currently, promising research in medicine is the search for pharmacological and molecular biological methods for activating HSP synthesis in cells.
Diseases associated with protein misfolding
Calculations have shown that only a small part of the theoretically possible variants of polypeptide chains can take on one stable spatial structure. Most of these proteins can take on many conformations with approximately the same Gibbs energy, but with different properties. The primary structure of most known proteins selected by evolution provides exceptional stability to a single conformation.
However, some water-soluble proteins, when conditions change, can acquire the conformation of poorly soluble molecules capable of aggregation, forming fibrillar deposits in cells called amyloid (from the Latin amylum - starch). Just like starch, amyloid deposits are detected by staining tissue with iodine.
This may happen:
1. with overproduction of certain proteins, as a result of which their concentration in the cell increases;
2. when proteins enter cells or form in them that can affect the conformation of other protein molecules;
3. upon activation of proteolysis of normal body proteins, with the formation of insoluble fragments prone to aggregation;
4. as a result of point mutations in the protein structure.
As a result of amyloid deposition in organs and tissues, the structure and function of cells are disrupted, their degenerative changes and proliferation of connective tissue cells are observed. Diseases called amyloidoses develop. Each type of amyloidosis is characterized by a specific type of amyloid. Currently, more than 15 such diseases have been described.