High pressure stabilization of collagen structure.

Scanning microcalorimetry has been used to study heat denaturation of small es, Cyrillicollagen at high pressure. It has been demonstrated that an increase of the pressure by 200 MPa enlarges the structure stability by 6.8 degrees C. The pressure increase does not affect the cooperativity of transition and only slightly decreases its enthalpy. The changes of the partial specific volume of collagen as well as its isothermal compressibility and thermal expansibility during transition have been estimated. In contrast to denaturation of most globular proteins, denaturation of collagen proceeds with an increase of the partial specific volume at ambient pressure. Moreover, the absolute value of the collagen volume increment is distinctly greater than this in most globular proteins. The volume increment diminishes when the pressure increases due to the difference in the compressibility for folded and unfolded states. This effect is compensated to some extent by the differences in heat expansion. The volume increment alters its sign when the pressure reaches about 324+/-20 MPa and the temperature of denaturation grows to 49.7 degrees C. Further pressure growing destabilizes the collagen structure.

[Does collagen microunfolding stimulate the fibril formation?].

The data on the effect of temperature on the kinetics of collagen fibril formation at physiological pH values and ionic strength in the temperature range 26-39 degrees C have been analyzed. The temperature of 35 degrees C optimal for collagen fibril formation has been defined as the turning point for halfmaximal turbidity and collagen molecule microunfolding values, which corresponds to the temperature of the first transition on the heat absorption curve. The temperature range (32-35 degrees C) in which collagen microunfolding stimulates fibril formation has been determined.

[Structural changes in wild-type Cry3A delta-endotoxin and its mutants in ethyl alcohol solutions at pH 2-2.5].

The methods of circular dichroism, scanning microcalorimetry, electron microscopy, and proteolysis were used to study the ability of wild-type Cry3A-delta-endotoxin and three mutant toxins with cysteine substitutions in helices alpha3 and alpha4 (domain I) to form oligomeric structures in acidic alcohol solutions that reproduce the premembrane environment. At pH 2-2.2 and 20% ethanol, the mutant toxins with single substitutions E132C (alpha3) and E160C (alpha4), as well as the double mutant E132C/S160C with a cysteine bridge connecting helices alpha3 and alpha4, form short linear oligomers specific for Cry3A with a high content of the beta-structure and enhanced sensitivity to proteolysis with pepsin. The data obtained show that the formation of oligomeric structures of this type does not require any divergence of helices alpha3 and alpha4 in domain I of the Cry3A toxin. It has been demonstrated that, at higher pH values in 20% solution of ethanol, the proteins studied are in a metastable state, and their ability to form oligomeric structures depends on temperature.

[On the mechanisms of the influence of imino acids on the physical characteristics of collagens].

The influence of imino acids on the thermodynamic characteristics of collagen type structures in various collagens has been analyzed. It was shown that the basic mechanism of entropy increase in the protein-water system consists in the alteration in the number of cooperative segments accompanying the increase in imino acids content, which can be observed during the melting of the fibrous macromolecule. The range of variation in the physical characteristics of cooperative units is determined, in particular, by the variability of hydrogen bond parameters. This is displayed in a broadening of the bands of NH-valence vibrations and the half-widths of transitions in post-denatured structures. Thus, the basic mechanism of the influence of imino acids on thermodynamic characteristics of collagens is related to the complex nature of the melting process. The dehydration-hydration mechanisms of native and denatured states become significantly different upon replacement of any amino acid by an imino acid.

[Thermodynamic characteristics of collagen fibrils reconstructed in vitro at different temperatures and concentrations].

The results of a calorimetric study of type I collagen fibrillogenesis were analyzed. The dependence of the half-width of the temperature transition of a collagen solution on the concentration and temperature of collagen formation was studied. It was demonstrated that, by varying temperature and collagen concentration, one can regulate the density of packing and dimensions of cooperative fibril blocks. At temperatures below the physiological level (25 degrees C and 30 degrees C), and a relatively low concentration of collagen (0.3 mg/ml), fibrils with the lowest density of packing are formed. The degree of order does not change as the collagen concentration increases twofold but grows as the concentration increases fourfold. It was shown that, at the physiological temperature (35 degrees C), fibrils with a dense packing of molecules are formed at all collagen concentrations studied. The value of fibril formation enthalpy is minimal at a temperature of 35 degrees C, pH 7.2, an ionic strength of 0.17 M and a concentration of 1.2 mg/ml. Based on the results obtained, a conclusion was made that the packing density of fibrils formed at physiological temperature does not depend on collagen concentration over the concentration range of 0.3 - 1.2 mg/ml.

[Structural and thermodynasmic aspects of the packing of collagen fibrils].

Studies devoted to the analysis of the mechanisms of packing of collagen fibrils and the effect of the macromolecular structure and the physicochemical parameters of medium on the packing are reviewed.

Heat capacity and conformation of proteins in the denatured state.

Heat capacity, intrinsic viscosity and ellipticity of a number of globular proteins (pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, myoglobin and cytochrome c) and a fibrillar protein (collagen) in various states (native, denatured, with and without disulfide crosslinks or a heme) have been studied experimentally over a broad range of temperatures. It is shown that the partial heat capacity of denatured protein significantly exceeds the heat capacity of native protein, especially in the case of globular proteins, and is close to the value calculated for an extended polypeptide chain from the known heat capacities of individual amino acid residues. The significant residual structure that appears at room temperature in the denatured states of some globular proteins (e.g. myoglobin and lysozyme) at neutral pH results in a slight decrease of the heat capacity, probably due to partial screening of the protein non-polar groups from water. The heat capacity of the unfolded state increases asymptotically, approaching a constant value at about 100 degrees C. The temperature dependence of the heat capacity of the native state, which can be determined over a much shorter range of temperature than that of the denatured state and, correspondingly, is less certain, appears to be linear up to 80 degrees C. Therefore, the denaturational heat capacity increment seems to be temperature-dependent and is likely to decrease to zero at about 140 degrees C.

[Model phospholipid membranes affect the holomyoglobin structure: conformational changes at pH 6.2].

The influence of model negatively charged membranes on the sperm whale holomyoglobin structure at pH 6.2 has been investigated by different techniques (far and near UV circular dichroism, tryptophan fluorescence, absorbance at Soret band, differential scanning microcalorimetry and fast performance liquid chromatography). It is shown that the holomyoglobin structure undergoes a conformational transition from the native to intermediate state analogous to its apo-form. This state is characterized by the absence of a rigid tertiary structure and the native heme environment. At the same time, the content of alpha-helical secondary structure remains almost native. To change the holomyoglobin structure similarly to that of its apo-form in the presence of membranes, a higher molar phospholipids/protein ratio is required. The properties of holomyoglobin in the presence of negatively charged membranes resemble those of the molten globule state of its apo-form protein in aqueous solution. A possible functional role of the discovered non-native myoglobin state is discussed.

[Structural changes in Cry3A delta-endotoxin from Bacillus thuringiensis var. Tenebrionis in alcohol solutions at pH 2.0].

Conformational changes in Cry3A delta-endotoxin caused by three different alcohols (ethanol, butanol, and isopropanol) were studied using the methods of circular dichroism, scanning microcalorimetry, and electron miscroscopy. It was shown that, in addition to the standard decrease in the native structure stability, the alcohols can cause a conformational transition that results in a sharp increase in the beta-structure content and a change in the environment of aromatic residues. The conformational transition is accompanied by intermolecular association, which leads to the appearance of oligomers in the form of short filaments. When the alcohols were removed, the oligomers dissociated again into monomers, but it is likely that the native structure either is not restored or is restored only in a small portion of molecules. The oligomer structure is rather cooperative, and its thermostability is higher than that of the initial structure. The disruption of this structure upon heating, observed as a heat absorption peak, is reversible.

Heat denaturation of ribonuclease.

One of the main and, chronologically, perhaps one of the first questions in the study of globular protein heat denaturation is that of the applicability of the "all or none" principle to this process, i.e., whether the transition of globular protein from the native into the denaturated state occurs abruptly, without intermediate, thermodynamically stable forms or there are several successive transitions. Despite an intensive study of the process of denaturation this question still remains unsettled. Moreover, its actuality has greatly increased lately with the accumulation of contradictory data.

Temperature-induced phase transitions in proteins and lipids. Volume and heat capacity effects.

The partial specific heat capacity and volume of globular proteins and dispersions of phosphatidylcholines in aqueous solutions have been determined over a broad temperature range using a precise scanning microcalorimeter and a vibrational densimeter. It is shown that the temperature-induced, gel-to-liquid crystalline phase transition in phosphatidylcholines proceeds without a noticeable change in heat capacity but with a significant increase in the specific volume, whereas heat denaturation in proteins takes place without a noticeable change in the volume but with a significant increase in heat capacity. This principal difference between temperature-induced conformational phase transitions in proteins and lipids demonstrates clearly that heat denaturation of proteins, in contrast to the gel-to-liquid crystalline phase transition in lipids, cannot be regarded as a process similar to melting. Consequently, the 'molten globule' does not appear to be a suitable model for a heat-denatured protein.

[Number of hydrogen bonds in the structure of collagen]. / Kolichestvo vodorodnykh sviazei v strukture kollagena.

The kinetics of hydrogen exchange of collagens from different animals was studied by the radioisotopic method (tritium) and infrared spectroscopy (deuterium). It has been shown that collagens from different animals (rat, pike, cod, carp, frogs) differ in amino acid composition and thermostability but are similar in the amount of slowly exchanged hydrogens. All the studied collagens have (1.00 +/- 0.05) very slowly exchanged hydrogens per triplet and (0.6 +/- 0.1) slowly exchanged hydrogens per triplet. Identifying the quantity of slowly exchanged hydrogens with the quantity of hydrogen bonds in the macromolecule, it can be concluded that collagens differing in stability do not differ by the quantity and composition of intramolecular hydrogen bonds.

[Conformational status of apomyoglobin in the presence of phospholipid vesicles at neutral pH]. / Konformatsionnoe sostoianie apomioglobina v prisutstvii fosfolipidnykh vezikul pri neitral'nykh pH.

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.

[Microcalorimetric studies of temperature transitions in fibrinogen and its proteolytic fragments]. / Mikrokalorimetricheskie issledovaniia temperaturnykh perekhodov v fibrinogene i ego proteoliticheskikh fragmentakh.

Melting of fibrinogen and its proteolytic fragments has been studied by differential scanning microcalorimetry. It has been shown that the fibrinogen molecule contains at least nine cooperative regions. One of them pertains to the central structural block (domain E), two of them are formed by the structures removed at proteolytic fragmentation and the others make part of the terminal structural blocks (domains D). A scheme of localization of melting regions in the fibrinogen molecule is proposed on the basis of the results obtained.

[Mobility of collagen structure and temperature adaptation of animals]. / Podvizhnost' struktury kollagena i temperaturnaia adaptatsiia zhivotnykh.

The method of hydrogen exchange is used to determine the mobility of acid-soluble collagen molecules from animals with different physiological temperature in terms of equilibrium constants of the formation of micro-unfolding or fluctuating defects of the structure. It has been shown that mobility of the collagen structure correlates with the physiological temperature of the animal from whose tissue collagen was isolated and is mainly determined by the amino acid content of the collagen. It has been shown also that at physiological temperatures characteristic for a particular species the level of collagen mobility is of the same order despite their different thermostability. The conclusion has been drawn that the level of mobility is the main criterion at the natural selection of the amino acid composition of collagens in different animals.

[Conformational states of the water-soluble fragment of cytochrome b5. I. pH-induced denaturation]. / Konformatsionnye sostoianiia vodorastvorimogo fragmenta tsitokhroma b5. I. pH-zavisimaia denaturatsiia.

Cytochrome b5 is a membrane protein that comprises two fragments: one is water-soluble and heme-containing, and the other is hydrophobic and membrane-embedded. The function of electron transfer is performed by the former whose crystal structure is known; however, its conformational states when in the membrane field and interacting with other proteins are still to be studied. Previously, we proposed water-alcohol mixtures for modeling the effect of membrane surface on proteins, and used this approach to study the conformational behavior of positively charged cytochrome c as well as relatively neutral retinol-binding protein also functioning in the field of negatively charged membrane. The current study describes the conformational behavior of the negatively charged water-soluble fragment of cytochrome b5 as dependent on pH. Decreasing pH was shown to transform the fragment state from native to intermediate, similar to the molten globule reported earlier for other proteins in aqueous solutions: at pH 3.0, the fragment preserved a pronounced secondary structure and compactness but lost its rigid tertiary structure. A possible role of this intermediate state in cytochrome b5 functioning is discussed.

[Microcalorimetric study of the domain organization of serum albumin]. / Mikrokalorimetricheskoe issledovanie domennoi organizatsii syvorotochnogo al'bumina.

Scanning microcalorimetry was used for studying the melting of the structure of human and bovine serum albumins and their fragments. It was shown that the melting of the native structure of serum albumin observed by the excessive heat absorption is a complex process which is described by three simple transitions overlapping in temperature. This means that the serum albumin molecule consists of three more or less independent cooperative structures, domains.

[Collagen denaturation enthalpy--nonlinear function of 4-hydroxyproline]. / Ental'piia denaturatsii kollagena--nelineinaia zatukhaiushchaia funktsiia 4-oksiprolina.

The results of calorimetric measurements of denaturation of collagens with different imino acid content are reported. In contrast to the existing point of view that denaturation enthalpy is a linear function of 4-oxyproline content, a nonlinear dependence was revealed. It is suggested that the reason for the observed nonlinearity is triplets of the (Gly-Pro-Hyp) type. An increase of their content can cause a decrease in the denaturation enthalpy in accord with the water-bridge structure and due to the minimum enthalpy effect of stabilization of the triplets as compared to triplets of other type.

[Temperature changes in ribonuclease]. / Issledovanie temperaturnykh izmenenii ribonukleazy

Deuteroexchange kinetics of ribonuclease is studied by the change of IR-spectra in the range of 1400--1900 cm-1 at different temperatures in solutions with two pD values, 2.6 and 5.1. It is shown that in the pre-denaturational temperature range the enthalpy of unfolding is 1.5 kcal/mol, i. e. two orders lower than the enthalpy of unfolding in the denaturational temperature range (100--120 kcal/mol). Thus in the pre-denaturational range of temperatures the disclosure of a compact protein structure can proceed at the expense of separate non-cooperative disruptures of bonds. However, the concentration of these disruptures is so low that they cannot give an explanation to the observed pre-denaturational macroscopic changes of protein.

[Thermodynamic study of guanylspecific ribonuclease C2]. / Termodinamicheskoe issledovanie guanilspetsifichnoi ribonukleazy C2.

Guanylspecific RNAse C2 has been studied thermodynamically using a microcalorimetry technique. It is shown that at ph varying from 5,0 to 8,0 the RNAse C2 solution in a cacodylate buffer contains about 30% of monomeric and about 70% of dimeric forms of protein, each of them melting as a single cooperative system. Moreover, studies of the influence of phosphate ions have shown that they stabilize the system by 3-4 degrees and transform it into a monomeric form.