How protein structures are built |
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The study of biological structures, their composition and molecular organization, their specific activity has become the subject of molecular biology. The success of the latter is associated primarily with the decoding of the structure of nucleic acids and the nature of hereditary information. A nucleic acid molecule is a linear sequence of four types of nucleotides arranged in a complex but strictly defined order, which can be compared with the regular arrangement of letters in a meaningful text. Just as a text carries some message, some information, the order of nucleotides in a nucleic acid molecule contains information about the individual structures of proteins that are to be created in the process of building an organism. A protein molecule is also a linear sequence of structural elements, but not nucleotides, but twenty types of amino acids. Each combination of three nucleotides in a nucleic acid molecule (genetic code) predetermines the inclusion of one or another of the twenty amino acids. The sequence of nucleotide triplets determines the exact sequence of amino acids in the synthesized protein molecule. Continuing the already generally accepted comparison of genetic information with written text, we can say that during protein synthesis, the text written in the nucleotide language is translated into the language of amino acids. The information contained in the amino acid text of a particular type of protein - that is, the composition and sequence of amino acids inherent to it alone - determines its shape and subtle internal organization - the spatial ordering of structural elements, on which certain of its biological functions depend. If this ordering is disturbed, enzyme proteins, for example, lose the ability to catalyze reactions in the body. Studies have shown that certain functions of a protein are directly performed by associations of chemical groups located in certain parts of an ordered protein molecule - specific functional centers. When the order is disrupted - for example, a protein molecule melts - then the combinations of chemical groups get the opportunity to change their mutual arrangement, scatter and functional centers cease to exist. Thus, the translation of the nucleotide language into the language of amino acids is not just a translation. Amino acid letters are much richer in physical and chemical content than nucleotide ones. And in general, the information carried by a protein molecule is fundamentally different from the nucleotide information, since it also determines the specificity of the structure of protein molecules and their subtlest biological functions. One more comparison can be made from the technical field. The information contained in nucleic acids is like blueprints from which parts are manufactured and assembled in a specific order. A protein molecule is an assembled mechanism, and the information contained in the sequence of its amino acids is the program of the mechanism itself. In a living cell, most proteins function not in a free state, but as components of complex structures — well-balanced and controllable systems, where each protein has a certain place and a certain share in the overall physiological function. The construction of complex cell structures is a dialectical transition from the field of chemistry (which should include the functioning of individual protein molecules) to the field of biology. Complex biological structures, in addition to proteins, also contain lipids, carbohydrates and other substances.However, in the construction of complex intracellular structures, the role of these substances is not the leading one. By the very nature of their chemical structure, carbohydrates and lipids simply cannot contain that very large amount of information that is necessary for such a construction. The most important role in it belongs to specific proteins. Thus, today's molecular biology confirms and details the well-known position of F. Engels about proteins as the basis of life. In proteins, where infinitely diverse molecules are built from structural elements with very different properties, where the precision of a unique organization is combined with flexibility and plasticity, nature has found an exceptional material that made it possible to create a higher, biological form of matter movement. The presence of specific centers is a common property of proteins that perform specialized biological functions. These are the "working organs" of protein molecules. Thanks to special specific centers, enzyme proteins selectively bind substances, the catalysts of chemical transformations of which are antitoxin proteins, bind toxins, etc. A system of interactions is organized between the chemical groups of a specific center and a partner molecule upon contact. It includes, firstly, electrostatic attraction between groups with opposite electric charges; second, the so-called hydrogen bonds between electrically polar groups; and, finally, third, "hydrophobic" bonds - interactions between non-polar groups (groups repelled by water). As a rule, stable chemical bonds do not arise here, since each individually of the listed interactions is rather weak. But in general, the system of a specific center provides sufficient strength of the connection of molecules. The above-mentioned selectivity of the action of specific centers is achieved due to the correspondence in the composition and arrangement of chemical groups in the very center and in the partner molecule - the so-called complementarity. Any replacement or movement of groups means a violation of the complementary ™. It is also clear that a specific center is not only a working mechanism, but also a cipher that allows a protein molecule to “recognize” its partner among many other molecules, even those with great similarity to this partner. The concept of specific centers reflects only the general character of the functional mechanisms inherent in proteins. The specific functions of proteins, the structure and reactions of their specific centers remain an area of science where almost everything remains to be done. This also applies to the processes of formation of supramolecular biological structures. Some biological structures are extremely complex. Such are, for example, membranes with * enzymatic complexes. The assembly of such structures is carried out, as the data of other studies show, by a large system of numerous protein components.The participation of many proteins in this work is, apparently, only indirect - they only participate in the process of creating a structure, but are not included in its composition. It is assumed that there are specific enzymes among these accessory proteins. On the other hand, there are biological structures that have a relatively simple structure. For example, other fibrous structures are built from protein molecules of only one type. In a number of cases in laboratories it is possible to decompose simple biological structures into their individual elements - protein and other molecules. Under appropriate environmental conditions, these elements are again combined in the right order and recreate the original structure. This re-creation process is commonly referred to as self-assembly. A number of research teams both abroad and in our country are studying its mechanisms. One such group is the Laboratory of Protein Structures and Functions of the Institute of Biochemistry, where self-assembly of fibrin fibers is studied. In favorable conditions for the body in the blood circulating through intact vessels, there is a soluble precursor of fibrin - the protein fibrinogen. When blood vessels are damaged, a special complex system of proteins begins to produce the enzyme thrombin, which cleaves four small particles called fibrin peptides from a large fibrinogen molecule. Having lost them, fibrinogen turns into fibrin-protein, the polymerization (connection with each other) of the molecules of which forms fibers. Monomeric fibrin molecules polymerize with strict ordering, which is characteristic of all self-assembly processes. Experimental studies of self-assembly processes require solutions Therefore, the first problem that arises before scientists who embark on the study of self-assembly processes is precisely the "dismantling" of biological structures. In each individual case, one has to look for methods of action specific to each structure that would effectively break the bonds between its constituent monomers and would not cause any harm to the monomers themselves. For fibrin, it was not possible for a long time to find a completely satisfactory way of decomposition of its polymer fibers. The solutions of urea initially proposed for this purpose and then of sodium bromide were ineffective. Only in 1965, an employee of our laboratory T.V. Varetskaya developed a method that completely satisfies all the requirements based on the use of dilute solutions of acetic acid at temperatures close to 0 ° C. The monomeric fibrin molecules obtained in this way always have the same properties, reproduced from experiment to experience. The previous methods of decomposition of fibrin in solutions of urea or sodium bromide did not give such constancy of properties: different samples of the monomeric protein obtained with their help differed, for example, in different rates of polymerization. Interestingly, when another protein, the structural protein of mitochondria, is obtained in a dissolved state, the best results (as concluded by American scientists studying the self-assembly of these structures) are also obtained by a cooled dilute solution of acetic acid. The processes involved in the self-assembly of structures are studied in various ways.One of these ways is a systematic study of the results of influencing the process of certain substances. For example, a delay in fibrin polymerization can be caused by exposing the starting monomer solution to an aqueous solution of inorganic salts, in particular sodium chloride. Within the limits of low salt concentrations - up to 2-3% - the delay in polymerization is the stronger, the "stronger" the solution. What information does this fact provide? It is known that salts in an aqueous solution exist in the form of ions carrying positive and negative electrical charges. The electrostatic efficiency of salt ions is usually estimated by a special value — ionic strength, which takes into account the concentration of the solution and the magnitude of the charge of its ions. The chemical nature of the individual salt ions is irrelevant here. The delay in polymerization is mainly determined by the ionic strength of the salt solution added to the monomeric protein solution. This shows that the effect is predominantly electrostatic in nature. Obviously, salt ions screen ("quench") the electric charges of monomeric fibrin molecules - a circumstance that just indicates that their electric charges are involved in the mechanism of selective connection of protein molecules. Under normal conditions - in the absence of interference from electrostatically charged salt ions - positively and negatively charged ionic groups, complementary located in specific centers, should attract molecules to each other. More detailed studies carried out in our laboratory by E.V. Lugovskii have shown that, along with the general screening effect of ionic strength, there is another effect of salts, which strongly depends on the chemical nature and individuality of ions and is determined by their ability to attach to a protein. The attachment of an ion to a specific center apparently introduces an additional disturbance in its work. E. V. Lugovskii investigated the effect of higher salt concentrations on polymerization. It turned out that some salts sharply delay, while others, on the contrary, accelerate polymerization. So, for example, two related salts, sodium chloride and bromide, act oppositely: the first accelerates, and the second retards the process. Like bromide, but even stronger, sodium iodide acts, like chloride, with different strengths - sometimes stronger, then weaker - sulfates, phosphates and some other salts act. It turned out that by the strength of the accelerating effect on fibrin polymerization, the salts are arranged in a row, which coincides with the long established and well-known series for the "salting out" (precipitation) of proteins in solutions with high salt concentrations. However, in experiments with fibrin polymerization, real salting out does not yet occur, since the process is studied at salt concentrations that still do not reach salting out ones. In addition, when salting out, proteins are precipitated in the form of a shapeless mass, and in the described case, normal fibrin fibers were formed - they could be seen using a phase contrast microscope. Many studies have found that the propensity of a protein to salting out is enhanced by the presence in its molecules of non-polar groups close to its surface and in contact with the environment. The more such groups, the lower the concentration of the saline solution, sufficient for salting out the protein. These well-known positions can be used to explain the results of our experiment, in which, undoubtedly, a salting-out effect is manifested, indicating that a monomeric fibrin molecule should contain a large number of non-polar groups on its surface. But we do not have real salting out. The salting-out effect is manifested only in the acceleration of specific polymerization. This can only be explained by the fact that non-polar groups are complementary components of a specific center of the protein molecule. Thus, studies of the effect of saline solutions on fibrin polymerization show that both electrostatic interactions and “hydrophobic” interactions between non-polar groups are involved in the process of fibrin self-assembly. The data of other studies indicate that the third type of interactions between protein molecules is also involved - hydrogen bonds. Let us now turn to fibrinogen, the precursor of fibrin. Its molecules are also capable of polymerizing to form fibrin-like fibers. Therefore, fibrinogen monomers also have specific centers. However, their polymerization requires special conditions and, in particular, a high ionic strength of the solution. If shielding of electric charges retards fibrin polymerization, then, on the contrary, it is a prerequisite for combining fibrinogen monomers in the chain. But it follows that the location of electric charges in a specific center of the fibrinogen molecule is unfavorable for polymerization and it should be carried out only through the interaction of those chemical groups that have no electric charge. Fibrin peptides, with the cleavage of which the fibrinogen molecule becomes a monomeric fibrin molecule, carry negative electrical charges. Apparently, their removal is the factor that changes the system of charges in a specific center and creates complementarity. Interestingly, one of the types of bleeding, a severe hereditary disease, is caused by a mutational change in fibrinogen, in which this protein loses its positive charges near the points of cleavage of fibrin peptides. The latter, as in the normal case, are cleaved, but thrombin no longer causes activation of fibrinogen, (As the diagram shows, activation consists in the fact that a nearby positive charge of a specific center is released from the neutralizing effect of fibrin-peptide. If there is no such charge, then cleavage of fibrin peptide becomes meaningless: activation does not occur.) Certain fragments of fibrinogen or fibrin are characterized by defective specific centers, which, however, are capable of selectively interacting with monomeric fibrin. Such fragments can be obtained by the destruction of these proteins by enzymes. In experiments with them, it is easy to observe how active fragments interact with fibrin and disrupt the assembly of fibers. It is precisely such experiments - the production and study of active fragments - that our laboratory is currently engaged in. It is hoped that by studying the structure and selective reactions of these fragments, we will better understand how proteins themselves are built and function. The complementarity of ionic groups, which plays such an essential role in the self-assembly of fibrin, is, apparently, also important in the self-assembly of other biological structures. The share of the energy of electrostatic bonds in the total amount of the interaction energy of the connecting molecules is probably small. More essential for the connection of molecules are "hydrophobic" bonds. But ionic groups can speed up self-assembly. Electrostatic charges can interact over a relatively long distance. And it is their long-range action that makes it possible, probably, to "probe" the environment, to recognize the desired partner and to contact him in an oriented manner. This suggests that when assembling very complex structures, which takes place in several stages, specific enzymes, like thrombin, must also act.It is easy to imagine the following sequence of reactions: a precursor protein, intended, for example, to participate in two assembly reactions, is activated by the first enzyme and combines with a specific partner; this makes it available for the second enzyme and subsequent specific attachment of the second partner. It is possible that this is precisely the mechanism of organization of those biological structures, the complexity of which excludes the possibility of direct self-assembly. At the intermediate stages of the assembly of complex structures, enzymes can be not only activation tools. Their action can alter the general properties of proteins. For example, a certain protein, already “embedded” in a structure, can become an insoluble part of it, having lost, thanks to enzymes, a significant part of its hydrophilic components. Of course, such a scheme does not exclude others, implying the possibility of the existence of carrier proteins that deliver insoluble proteins to the assembly site. In conclusion, it should be noted that the study of the assembly processes of supramolecular biological structures is an area replete with unclear and complex questions. Therefore, at this stage of its development, information about the processes occurring in such relatively simple systems as the system of fibrin fiber formation is especially interesting and useful. V. Belitser Similar publications
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Modern biology has penetrated deeply into the depths of the cell — the “brick” of the living. A living cell appeared to scientists as a harmonious combination of simpler structures - membranes, tubes, granules, fibrous formations, consisting of ordered molecules connected to each other.
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