Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations

Thus, it is the denaturation of proteins that makes these polymers active. Their activity arises from the fact that only denatured proteins begin to interact with each other. This interaction seems to be specific and regular; native aggregation results in new structures that are absent in the resting state and have physiological meaning for the active state. In other words, denaturational changes make proteins reaction-capable. While these changes are reversible, the cell is able to disassemble the temporary structures formed and to return to the initial state - the resting state. When damage ensues, when protein aggregation becomes too extensive or irreversible, pathological changes appear in the cell and can lead to its death. The threshold character of the cellular reaction means that the resting state and the active state are different thermodynamic states of the system, which are separated by an energy barrier; this relates not only to the cell as a whole, but also to its individual components [5].

Now the time has come to ask: what makes protein aggregation specific? The answer to this question is provided by the physics of proteins. It has been established that the correct folding of a polypeptide to a globule, like the unique structure of the globule itself, is provided by specific interactions between protein secondary structures [7]. Let us consider a structure such as an α-helix. It interacts with other secondary structures via its surface. The surface of the α-helix is "encrusted" with polar (hydrophilic) and non-polar (hydrophobic) groups. Taken individually, these groups are capable only of nonspecific interactions, but the secondary structures confer a specific character on these interactions. This is their biological meaning. Indeed, depending on the amino acid composition, the topography of hydrophobic groups on the surface of an α-helix can vary strongly. If two α-helices have complementary topographies of hydrophobic amino acid residues, such secondary structures will "recognize" each other and associate to form a hydrophobic nucleus (the principle of "key"-to-"lock" correspondence works here, too). Owing to the same topographic factor, polar groups can form on the secondary structure surface a "landscape" complementary to the nucleic acid surface. To provide specificity of interaction by a unique distribution of protein functional groups on the surface is the main purpose of all protein secondary structures. The principle of structural complementarity has a universal physical basis and is realized not only in intraprotein interactions (in the globular proteins formed and in the process of their folding), but also in interprotein interactions (native aggregation) including protein-nucleic acid interactions.

When an action on a cell or cell structure exceeds the threshold, (i) formation of secondary structures begins in natively unfolded proteins (or unfolded regions of proteins), while (ii) secondary structures of molten globules start to become accessible for interaction with secondary structures of other proteins and with nucleic acids. Such secondary structures induced by the external action are centers (sites) of native aggregation. Thus, the first event in the activated cell is the appearance of new secondary structures able to interact selectively with each other to form tertiary, quaternary, etc. structures. Proteins whose secondary structures appear under such circumstances lose their previous inertia and become reaction-capable.

The proposed approach to understanding the mechanisms of cellular reactions poses the question of native and denatured protein states in a new way. In the native state the key cell proteins are inert, non-reaction-capable; they do not interact with each other or with other biopolymers. Loss of the state of inertia is denaturation. On denaturation of the unfolded polypeptide chains the secondary structures appear, whereas on denaturation of molten globules their secondary structures are modified and "float up" to the surface from the hydrophobic nucleus. In both cases the secondary structures are ready to interact. In other words, two extreme protein states can be identified: the completely folded (the globular protein) and the completely unfolded states. Between these inactive (native) states, numerous intermediate, active forms can exist; it is these forms that provide native aggregation. Thus, in proteins, only two states are inactive (they are native states). In all other cases they are active, as manifest in the capacity for native aggregation.

The proposed mechanism of native aggregation explains the increase of volume of the cellular hydrophobic phase during the protoreaction [8] and the structural changes in the universal reaction of the living cell [1]. When secondary structures form, the polar groups of peptide bonds break contact with water and form hydrogen bonds with each other. For this reason alone the hydrophobicity of a polypeptide with secondary structures is higher than in the unfolded polypeptide-precursor. The volume of the hydrophobic phase increases even more when the secondary structures fuse to form hydrophobic domains (nuclei). The second reason why the volume of the cell hydrophobic phase increases further is the appearance of molten globules. In native globular proteins the hydrophobic nucleus is a solid body with a comparatively small surface interacting weakly with hydrophobic substances (therefore, the cell in the resting state is hydrophilic). On melting, the hydrophobic nucleus ceases to be a solid body ([7], Lecture 17); its constituent elements become much more mobile relative to each other, and the nucleus loosens and becomes accessible to water and to substances dissolved in it (surface hydrophobic contacts increase). If the solution contains hydrophobic compounds, it becomes possible for them to penetrate into the molten globule nucleus and become concentrated in this hydrophobic phase.

Proteins in the excited state are capable not only of new intramolecular interactions, but also of interaction with other proteins. Protein physics offers no prohibitions on this point. Native aggregation (formation of specific aggregates) explains the increase of cell turbidity and of macroscopic viscosity of the cytoplasm and nucleus. Thus, the observed changes during the protoreaction are given a simple explanation based on data from protein physics [7].

In this section, significant attention was paid to the cell in the resting state. Let us now consider it in greater detail.

To study any process, it is important to identify a starting point. For instance, it would have been impossible to understand the mechanism of muscle contraction without the concept of the resting state of the contractile apparatus. Based on the experience of classical physiology, it is necessary to accept that the concept of the resting state of cell (as well as of its individual parts) is of great importance for understanding the mechanisms of activation. Here we return again to the issue of the structure of the resting cell. The fact that such a cell, unlike an activated one, is almost completely transparent, indicates a negligible amount of protein aggregate. Also, the resting cell is hydrophilic, as under conditions of diffusional equilibrium it does not bind vital dyes [1], which are hydrophobic [8]. These essential peculiarities of the resting cell are to be explained by its structure.

Ling [22] was the first to suggest that the structure of the resting cell is determined by natively unfolded proteins. This concept was finally formulated by 1965 [23], while a summary of the development of this way of thinking was published a decade later [6]. The most important argument in favor of this point of view is the identity of the equilibrium distribution of substances between the cell and the medium on the one hand, and between the model systems and the medium on the other. The model systems studied include cellophane dialysis bags filled with concentrated solutions of hydrophilic and electrically neutral linear polymers, all of whose chain links are accessible to water. The distribution law, i.e., dependence of equilibrium distribution of substances on their concentration in the medium, is the same for the model systems and for the living cell. Since the distribution of substances was studied under conditions of diffusional equilibrium, this result means that the key physicochemical factor determining the character of the distribution is identical in the models and the cell, and is provided by unfolded biopolymers. It seems obvious that of all cell polymers, only proteins - the most massive cell polymers - can possibly fulfill this role [23].

What is this factor? Both cells and models have a common peculiarity: if the solution component studied is not absorbed on a polymer within the system, its equilibrium concentration in the internal medium is always lower than in the external solution. Model systems, owing to their simplicity, allow this phenomenon to be understood: it is because substances are less thoroughly dissolved in the system water than in the water of the external medium. Physics provides the only possible explanation for this difference: water in the cell and in the model systems is more ordered than bulk water; therefore, insertion of a molecule of solute with more rigid bonds into the solvent is not energetically advantageous, so solutes are displaced (excluded) from the system. But why is water ordered in the presence of linear polymers? The obvious explanation is provided by model systems comprising nothing but polymer, water, and dissolved substance: if water is absorbed by the regularly repeated polymer links, the water itself is ordered in the space (multilayer adsorption). Also, in the absorbed water molecules, the electrical properties are different.

In spite of the wide diversity of proteins, they all have absolutely identical polypeptide backbones; differences between proteins are due only to the side chains. The polypeptide backbone of all proteins comprises a regular alternation of positive (NH) and negative (CO) charges in the peptide bonds; the distance between these groups turns out to be comparable with the size of a water molecule and with the length of the hydrogen bonds between them. In other words, the disposition of these dipoles along the polypeptide backbone is complementary to water structure. Another peculiarity of the peptide bond groups is that they form hydrogen bonds either with each other (in the secondary structures) or with water (in the unfolded regions of the polypeptide chain) ([7], Lecture 4). However, the question arises - why does the interaction of water with the functional groups of peptide bonds change its properties so markedly? To answer this question, let us address the properties of electric dipoles.

An important property of dipolar molecules is that their dipole moment is not constant, but depends on their interaction with other dipoles [24]. Example: the dipole moment of water in the gaseous phase is equal to 1.85 D, while in the liquid phase it is 2.9 D. Hence, the interaction of water molecules with each other leads to their mutual polarization - an enhancement of their own dipole moment by 60% [25]. But what if the water molecule interacts with a stronger dipole than itself? The dipole moment of a peptide group is 3.5 D [26]. If water interacts with these, stronger, dipoles, its molecules will be polarized to a greater degree and their hydrogen bonds with other molecules will become stronger. The enhancement of hydrogen bonds makes the first adsorptional layer stable and able to attract and to bind more and more new free water molecules, forming more and more new adsorbed layers. Thereby, stronger dipoles on the adsorbing surface are the key prerequisites for the multilayer adsorption of polar molecules.

Owing to the enhancement of hydrogen bonds in the multilevel adsorbed water layer, penetration of other molecules into it (including water itself) becomes energetically non-advantageous, because it requires breakdown of the intermolecular hydrogen bonds in the layer, which are stronger than in the voluminous (bulk) phase. This explains why bound water is a poor solvent compared with the phase in which water molecules interact only with each other. For this thermodynamic reason, the concentration of any substance in the absorbed phase always will be lower than in the liquid phase.

However, all begins to change if the unfolded polypeptide absorbing water begins to fold with formation of secondary structures. In this process, peptide groups cease to form hydrogen bonds with water and form them between each other. The previously bound water is desorbed and acquires the properties of voluminous (bulk) solvent [6,23,27]. There is convincing experimental evidence to substantiate this point of view about the interaction of polypeptides and other hydrophilic polymers with water [28,29].

But what is the role of globular proteins? It is these compounds that are the second important component of the cell in the resting state. They are the best-studied type of proteins, performing structural and enzymatic functions. Their solid core is inaccessible to water, while polypeptide chains containing no secondary structures are not sufficiently expanded to affect the state of the intracellular water fundamentally [5].

Thus, in the resting state, the physical properties of the cell protein matrix are determined by partially or completely unfolded proteins and by globular proteins (of course, the latter include complex proteins with several globular domains). In the context of the present paper, such proteins can meaningfully be called native. The structural and functional peculiarities of the cell in the resting state are determined by unfolded proteins [5].

The question remains as to why the resting state of the cell is relatively stable and can exist for an indefinite period. Ling believes this is accounted for by the stabilizing effect on unfolded proteins of various ligands bound to native unfolded proteins: ions, low-molecular organic compounds, hormones, etc. According to Ling, the most important ligand of proteins in the resting state is ATP [30]. If some action leads to splitting of ATP or to dissociation of other rest-making ligands, this leads to folding of the natively unfolded protein; secondary structures appear and make the polypeptide reaction-capable. Native aggregation begins, in the course of which signaling structures are formed. Natively unfolded proteins seem to be the most sensitive elements of the resting cell, as their folded state is economically advantageous, because when the water is desorbed the entropy of the system increases (water is the most abundant cell component). Also, the rest-making ligands are not firmly bound to natively unfolded proteins, as the bonds are non-covalent, while ATP can be split enzymatically. As a result, individual cell components or the entire cell appear as a system in which the structural content of life activity is the reversible transition from the resting state into the activated (excitatory) state provided by the reversible transition of proteins from the resting (native) into the activated (non-native) state.

From the point of view of the proposed approach, reactions of the cell to external actions, various forms of cellular activity (metabolism, division, muscle contraction, secretion, intracellular signaling, etc.) as well as pathological states are considered on the basis of the following statements and principles.

Native aggregation is a specific interaction of proteins with each other, realized by interaction between the secondary structures of the aggregating proteins. If the reaction-capable secondary structures are absent or inaccessible for interaction, native aggregation is impossible.

The cell is considered as a system that can have only two states: the resting state and the active (excitatory) state. The same principle is true for any cell organelle, structure or protein molecule. For clarity, a parallel can be presented: the excitable membrane in a state of rest or excitation.

Functionally important cell proteins in the resting state are present in one of two states: either unfolded (completely or partly - natively unfolded proteins) or folded to the protein globule state or any other form in which secondary structures are inaccessible for interaction with other proteins. These states are considered the resting states of protein molecules or as their native states. Proteins in the native state are stabilized by rest-making ligands and/or chemical modifications, for instance, by phosphorylation/dephosphorylation. According to Ling [30], the resting state of an unfolded protein is maintained by its bound ATP, ions (Na+, K+, Ca2+), molecules of bound water, hormones (for instance, insulin), and any other significant interactions. For instance, analysis of amino acid sequences in the regions surrounding known phosphorylation sites reveals a strong propensity towards adoption of a natively unfolded conformation [31]. Disruption of bonds with ligands (for instance, breakdown of ATP) leads to activation of the protein, its transition from the resting to the active state; the same result is produced by a decrease in the cell ATP content below the critical level. Ling's concept of the capability of small molecules for specific binding with natively unfolded proteins is confirmed, for instance, in the work by Mukhopadhyay et al. [32].

On activation of the cell by external actions, intracellular factors, and signals of different nature (including chemical modification), a new protein fraction appears -- activated proteins with newly formed secondary structures that were absent in the resting state (Fig. 2). These new structures appear on the folding of natively unfolded proteins and on melting of protein globules. They include α-helices, β-sheets, and other secondary structure variants. The secondary structures of activated proteins are new "valences" necessary for new interactions - intramolecular (folding) and intermolecular (native aggregation). In the case of large proteins, the secondary structures can form hydrophobic sites on the protein surface, which interact specifically with similar (complementary) structures on the surfaces of other proteins.

The natively unfolded proteins can be called excitable proteins. Their transition to the excitatory state triggers native aggregation.

If on the unfolding of a globule (or a globular domain) no molten globule intermediate is formed, while the protein cooperatively assumes the completely unfolded configuration at once, this means it is inactivated, as a protein without reaction-capable secondary structures is incapable of aggregation. The molten globule may be inactivated in two ways: by transition back to the well-folded conformation (when secondary structures are hidden from interaction) or by unfolding of the molten globules until a completely unfolded conformation is reached, devoid of the secondary structures that are key to native aggregation.

The secondary structures in activated proteins play the role of centers of native aggregation. It is these structures that provide for specific interactions of activated proteins with each other (native aggregation) to form new structures that have signaling and functional significance for the active cell. Native aggregation is determined by the same forces and interactions that are involved in the well-studied folding of unfolded polypeptides to globules. This rule is followed: if there are secondary structures capable of specific interaction, there is native aggregation; if there are no such structures or they are inaccessible, there is no native aggregation.

If an action on a protein increases the number of amino acid residues included in its secondary structures, that protein is activated and the signal pathways in which it participates are open. If the protein is unfolded and the portion of amino acid residues in secondary structures decreases, it undergoes transition to the inactive state, is relaxed, while the signal pathway(s) in which it participate(s) is/are blocked. On the transition of proteins participating in native aggregation to the native state, native aggregates are destroyed and individual structures and the cell as a whole transit to the resting state.

The temporary structures appearing as a result of native aggregation perform diverse functions. They may be centers of specific adsorption (binding) of various ions and molecules including signal factors and proteins, i.e., can perform the functions of receptors. They may have the enzymatic activity necessary for performing specific functions and may serve as centers of formation of even more complex supramolecular structures.

Only the secondary protein structures are able to provide for specificity (selectivity) in the interaction of proteins with others, as they provide the specificity of interactions necessary for correct folding of the polypeptide chain to a globule (the folding of polypeptide to native globule can be considered as intramolecular native aggregation). Each secondary structure has a unique topology of polar and hydrophobic groups on its surface. Secondary structures form stable complexes with each other or with sites on nucleic acids only if their surfaces are complementary to each other, as the key is complementary to the lock.

Native aggregation is determined genetically to the same extent as protein structure because it is determined by the same factors that determine all levels of organization of the individual protein molecule beginning with the primary sequence. Secondary structures of activated (excited) proteins will interact with other excited proteins not chaotically, but in accordance with the genetic program. As a result of native aggregation, those structures and corresponding functions will appear that are necessary to the cell here and now: action potential, channels on the cell surface, in the cytoplasm and nucleus, cytoskeleton, movement of cytoplasmic sites, cell division, apoptosis. Errors in native aggregation that appear during a prolonged state of cell excitation (for instance, chronic inflammation) and on damage lead to various forms of cellular pathology: conformational diseases, necrosis, carcinogenesis.

All the differences between the excited cell and the cell in the resting state are the direct or indirect results of native protein aggregation.