[Kinetics and mechanism of peptide synthesis in solution]. / Kinetika i mekhanizm sinteza peptidov v rastvore.

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied measuring changes in the fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data couldn't be described by a simple scheme of the second order reaction. The measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: v = kCNaCAEb, where k is the reaction rate constant, CN is the concentration of leucinamide, and LeuNH2, CAE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates. The English version of the paper.

[Point amino acid substitutions in Ca2+-binding centers of recoverin. III. Mutant with the fourth reconstructed Ca2+-binding site]. / Tochechnye aminokislotnye zameny v Ca2+-sviazyvaiushchikh tsentrakh rekoverina. III. Mutant s chetvertym rekonstruirovannym Ca2+-sviazyvaiushchim tsentrom.

Unlike wild type recoverin with only two (the second and the third) functioning Ca(2+)-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca(2+)-binding site. This site was reconstructed from the fourth potential Ca(2+)-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca(2+)-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca2+ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild type recoverin.

[Single amino acid substitutions in the Ca2+-binding site of recoverin.II.The unusual behavior of the protein upon the binding of calcium ions]. / Tochechnye aminokislotnye zameny v Ca2+-sviazyvaiushchikh tsentra rekoverina. II.Neobychnoe povedenie belka pri vzaimodeistvii s ionami kal'tsiia.

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca(2+)-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca2+ ions results in an increase in the mobility of the environment of Trp residues, in higher hydrophobicity, and in elevated thermal stability (its thermal transition shifts by 15 degrees C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca2+ are also characteristic of the -EF2 mutant of recoverin whose second Ca(2+)-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca(2+)-binding sites, respectively, are modified and damaged) are practically indifferent to calcium ions.

[Use of method of protein engineering in studying calcium-binding proteins]. / Issledovanie metodov belkovoi inzhenerii v issledovanii kal'tsiisvaiushchikh belkov.

Major results of the use of protein engineering methods in studies of calcium-binding proteins with the highest affinity for calcium and known three-dimensional structure (parvalbumin, calmodulin, troponin C, calbindin, recoverin, alpha-lactalbumin, and others) are presented. Specific features of recombinant calcium-binding proteins are discussed. Experiments with genetic introduction of fluorescent probes, tryptophan and tyrosine, into proteins are overviewed. Effects of mutations in different parts of protein molecules (calcium-binding loops, hydrophobic core, and others) on their structure and properties and attempts of creation of artificial calcium-binding sites are discussed.

[p-Sulfotetrafluorophenyl hydrophilic activated esters of amino acids in peptide synthesis]. / p-sul'fotetraftorfenilovye gidrofil'nye aktivirovannye éfiry aminokislot v peptidnom sinteze.

Highly reactive hydrophilic (i.e., water-soluble) p-sulfotetrafluorophenyl esters (Tfs esters) are proposed for peptide synthesis in aqueous and aqueous-organic media, as well as for protein and peptide partial synthesis in an aqueous medium. These esters can serve as a basis for creating a series of protein modifying reagents. As they are analogs of the widely used pentafluorophenyl esters, the Tfs esters possess a high reactivity coupled with good stability during storage. The expression for the reaction rate (for substrates AA1 and AA2) is shown to be v = k[Boc-AA1-OTfs][H-AA2-NH2]0.5 for both water and DMF, i.e., the reaction is not a simple second-order reaction. The reaction rate in water is only slightly lower than that in DMF.

[The effect of phalloidin on stability of F- and G-actin]. / Vliianie falloidina na stabil'nost' F- i G-aktina.

Intrinsic tryptophan fluorescence and fluorescence of a hydrophobic probe bis-ANS were used to study the effect of phalloidin, a bicyclic heptapeptide toxin, on stability of monomeric (G) and polymeric (F) actin. It was found that bis-ANS fluorescence is sensitive to the actin polymerization process. Phalloidin in concentrations from 1 to 2 molecules per 1 actin molecule shifts the thermally induced unfolding transition in F-actin toward about 15 degrees C higher temperatures. The stabilizing effect of phalloidin is even more evident in the case of urea denaturation of F-actin. Moreover, phalloidin stabilizes against denaturing by not only F-actin, but G-actin as well, showing direct stabilizing interactions between phalloidin and G-actin.

[Study of conformation transitions in proteins by tryptophan fluorescence and phosphorescence at low temperatures]. / Issledovanie konformatsionnykh perekhodov v belkakh po triptofanovoi fluorestsentsii i fosforestsentsii pri nizkikh temperaturakh.

The well-known conformational changes in proteins containing a single tryptophan residue, such as pH-induced N-->F transition in human serum albumin, pH-induced acidic transition in cod parvalbumin, and KCl-induced tetramerization of bee venom melittin were monitored by changes in low temperature phosphorescence and fluorescence spectra suggesting two independent series of normal components. Parameters of low temperature tryptophan luminescence were sensitive to chromophore environment. A correlation of changes of some spectral parameters with accessibility of tryptophan to water was revealed, however, spectral changes mainly depend on specific interactions of the chromophore with its environment.

[Analytical description of electron-vibrational protein spectra]. / Analiticheskoe opisanie élektronno-kolebatel'nykh spelktrov belkov.

Electron-vibrational spectra of phosphorescence and fluorescence of tryptophan residues in proteins at 77 K are best approximated by theoretical curves computed according to a model which suggests the existence of two independent series of Gaussian vibrational components. Each series contains one type of vibrations. Phosphorescence and fluorescence spectra of proteins with various localizations of their single tryptophan residue were fitted by a curve computed according to this model. The results obtained show that the phosphorescence band of tryptophan residues in proteins seems to contain two types of vibrations with frequencies 650-800 cm-1 and 1350-1500 cm-1. Since the substitution of H2O by D2O does not change the frequencies of both vibrations in the phosphorescence spectra of human serum albumin, melittin and tryptophan in 1 M KCl, it is reasonable to suggest that the 1350-1500 cm-1 series corresponds to the W5 type vibrations (B19a type of vibrations of benzene ring). The 650-800 cm-1 series"can be identified with W18 type of vibrations (breathing vibrations of indole ring). Phosphorescence parameters of tryptophan residues in proteins correlate with their fluorescence parameters.

[Fluorescence monitoring of solid-phase peptide synthesis using a quartz reactor-cuvette]. / Fluorestsentnyi kontrol' tverdofaznogo sinteza peptidov s primeneniem kvartsevogo reaktora-kiuvety.

The quartz reactor-cuvette for fluorescent monitoring of the solid phase peptide synthesis consists of a 10 mm diameter quartz tube jacketed in a 14 x 14 mm orthogonal holder and equipped with chemically inert adapters. This allows to use the reactor with almost any standard fluorimeter. Details of the construction are presented. Fluorescence spectra of Fmoc-group, both in solution and on the solid support, are given; effects of concentration quenching of the fluorescence are studied.

[Study of thermal denaturation of the rod part of myosin molecule by microcalorimetry and intrinsic fluorescence methods]. / Issledovanie teplovoi denaturatsii sterzhnevoi chasti molekuly miozina metodami mikrokalorimetrii i sobstvennoi fluorestsentsii.

Thermal denaturation of myosin rod has been studied by differential scanning microcalorimetry and intrinsic tryptophane fluorescence methods. Use of the sequence annealing in the calorimetric measurement allows to decompose the total thermogram of rod into four elementary bands with maxima at 42, 46.5, 50 and 57 degrees C. Fluorescence changes occur at temperatures which coincide with the first, second and fourth calorimetric peaks. Changes of the time resolved and steady state fluorescence of myosin rod were interpreted using the data on localization of tryptophan residues in the molecule. The tryptophan fluorescence of myosin rod is assumed to monitor the denaturational changes in high meromyosin and probably in the hinge region but not in the subfragment 2.

[Ca-inhibited binding of melittin with parvalbumin. A new role for parvalbumins?]. / Ingibiruemoe ionami Ca sviazyvanie melittina parval'buminom. Novaia rol' dlia parval'buminov?

It was found that pike parvalbumins pI 4.2 and 5.0 bind amphiphilic peptide melittin extracted from bee venom in an extraordinary Ca-dependent manner: in apo-state the protein forms a tight equimolar complex with melittin (Ka = 10(6) M-1 at 18 degrees C); in Ca- (and Mg-) loaded state it does not take place. Heating of the protein up to temperatures above the denaturation temperature of apo-parvalbumin does not change the stoichiometry of the complex but increases its association constant by an order of magnitude (Ka = 1.2.10(7) M-1 at 44 degrees C). Isolated Ca-binding domain of parvalbumin, 38-108, retains the ability for Ca-inhibited binding of equimolar quantities of melittin. The possible function of parvalbumin in vivo is suggested: Ca-inhibited interactions with some intracellular components.

[Effect of temperature and pH on the environment of tryptophan residues in alpha-actinin]. / Vliianie temperatury i pH na okruzhenie ostatkov triptofana v alpha-aktinine.

Effects of temperature and pH on the structure of rabbit muscle alpha-actinin were studied by means of an intrinsic fluorescence method. Alkaline denaturation of alpha-actinin at 15 degrees C begins at pH above 9, while acidification of the solution does not cause unfolding of the protein structure, but results in protein aggregation. The maximal intensity of the isoelectric aggregation process is registered at pH 5. Thermal denaturation of alpha-actinin occurs within the temperature range from 45 degrees C to 65 degrees C. Protein has the second thermally induced transition in the region from 17 to 30 degrees C.

[Interaction of copper nd zinc cations with calcium-binding proteins]. / Vzaimodeistvie kationov medi i tsinka s kal'tsiisviazyvaiushchimi belkami.

Interactions of the calcium binding proteins, parvalbumin from cod muscles, alpha-lactalbumin from cow milk and calmodulin from bovine brain, with Cu2+ and Zn2+ ions have been studied by intrinsic fluorescence and microcalorimetry methods. It was revealed that parvalbumin binds one Cu2+ ion per molecule with association constant from 10(5) to 10(6) M-1. Zn2+ ions seem to compete for the same site which does not coincide with the two Ca2+ and Mg2+ binding sites. alpha-Lactalbumin contains from 2 to 4 Cu2+ and Zn2+ binding sites, the number and affinities of which depend on Ca2+ concentration. Calmodulin has similar Cu2+ and Zn2+ binding sites. The binding of Cu2+ and Zn2+ ions to parvalbumin and alpha-lactalbumin changes the shape and position of their thermal denaturation transitions. The results obtained together with the literature data show that the ability to interact with Cu2+ and Zn2+ ions is a property inherent to many calcium-binding proteins, which may play a physiological role for some of them.

[Kinetics of dissociation of alpha-lactalbumin complexes with Ca2+ and Mg2+ ions]. / Kinetika dissotsiatsii kompleksov alpha-laktal'bumina s ionami Ca2+ i Mg2+.

Kinetics of dissociation of the complexes of bovine alpha-lactalbumin with Ca2+ and Mg2+ ions induced by mixing of the Ca2+- or Mg2+-loaded protein with the chelator of divalent cations EDTA has been studied by means of intrinsic fluorescence stopped flow method. Within the temperature region from 10 to approximately 37 degrees C the fluorescence kinetics curves for the Ca2+ removal are well fitted by one exponent with the rate constant ranging from 6.10(-3) to 1 s-1. Taking into account rather low rate of the fluorescence changes, one can assume that the limiting stage in this case is the dissociation of the single bound Ca2+ ion from the protein but not a conformational change which occurs after the Ca2+ dissociation. At temperatures above 37 degrees C the kinetics curves are best fitted by two exponents. The second exponent seems to be due to the denaturation of the apo-form of alpha-lactalbumin which takes place at these temperatures. The values of the dissociation rate constants of Mg2+ practically coincide with the values for Ca2+.

[The effect of calcium and magnesium ions on the interaction of calcium-binding proteins parvalbumin and alpha-lactalbumin with dipalmitoylphosphatidylcholine vesicles]. / Vliianie ionov kal'tsiia i magniia na vzaimodeistvie kal'tsiisviazyvaiushchikh belkov parval'bumina i alpha-laktal'bumina s vezikulami dipal'mitoilfosfatidilkholina.

Interactions of the calcium binding proteins, like parvalbumins pI 4.2 and p15.0 and bovine and human alpha-lactalbumins, with dipalmitoylphosphatidylcholine vesicles have been studied by means of scanning microcalorimetry and intrinsic tryptophan, tyrosine and phenylalanine fluorescence. The interactions are modulated by the Ca2+ and Mg2+ binding to the proteins and induce some changes in the physical properties of both the proteins and the liposomes. The liposomes increase the thermal stability of the Mg2+-loaded and metal-free parvalbumin. Ca2+-loaded alpha-lactalbumin interacts with the liposomes in its native state, while the metal-free protein binds to the liposomes mainly in its thermally denatured state. The interactions of both proteins with the liposomes affect the phase transition from gel to liquid-crystalline state in the liposomes. The results of the microcalorimetric and spectrofluorometric studies are corroborated by the data obtained by means of gel-chromatography on Sepharose 4B.

[Study of the physico-chemical properties of troponins I and T from the heart and skeletal muscles using protein fluorescence and calorimetry methods]. / Issledovanie fiziko-khimicheskikh svoistv troponinov I i T iz serdechnykh i skeletnykh myshts metodami belkovoi fluorestsentsii i kalorimetrii.

Physico-chemical properties of troponin I and troponin T subunits from cardiac and skeletal muscles were studied, using intrinsic protein fluorescence and differential scanning microcalorimetry. The effects of temperature, pH, urea and ionic strength were analyzed. Similar skeletal and cardiac components were shown to possess similar properties. Alkali produced structural changes in both troponins I which seems to be initiated by deprotonation of histidyl side chains within the pH range of 6.5-9.0. An increase of pH from 9 to 12 results in alkaline denaturation transitions in both troponin I subunits, which might be due to deprotonation of tyrosyl side chains. A decrease of pH from 6 to 4 causes aggregation of both troponin T subunits. Cardiac troponin T is more stable to alkali and urea denaturation than the skeletal one. Heating up to 100 degrees C does not cause any cooperative denaturation transitions in troponins I and troponins T. These results suggest that cardiac and skeletal troponins I and troponins T possess a rather open, not highly ordered structure in solution.

[Relation between the thermostability of the tetrameric molecule of lactate dehydrogenase from swine muscles and the degree of occupancy of its active sites with ligands]. / Vzaimosviaz' mezhdu termostabil'nost'iu tetramernoi molekuly laktatdegidrogenazy iz myshts svin'i i stepen'iu zaniatosti ee aktivnykh tsentrov ligandami.

Using differential scanning microcalorimetry and measurements of protein fluorescence, the thermal denaturation of lactate dehydrogenase (LDH) from porcine muscle (in the apo-form as well as in the form of the enzyme-pyruvate, enzyme-NAD+ and enzyme-NAD-pyruvate-adduct complexes) was studied. Pyruvate binding did not affect the thermal stability of LDH. NAD+ exerted a stabilizing effect on the enzyme, the value of which was proportional to the number of ligand molecules bound per LDH tetramer. The formation of the abortive LDH-NAD-pyruvate complex in one, two or three active centers of the enzyme tetramer did not influence the values of calorimetric parameters of thermal denaturation in comparison with those for the apoenzyme. The occupancy of all four active centers of LDH by the adduct resulted in a sharp increase of the enzyme thermal stability and tightness of the LDH adduct complex as compared with complexes formed upon partial saturation. The experimental results are suggestive of the existence of a concerted conformational transition of the LDH tetramer induced by the formation of the LDH-NAD-pyruvate complex in the last active center of the tetramer.

[Kinetics of dissociation of parvalbumin complexes with calcium and magnesium ions]. / Kinetika dissotsiatsii kompleksov parval'bumina s ionami kal'tsiia i magniia.

Dissociation kinetics of parvalbumin complexes with calcium and magnesium ions were studied by means of stopped-flow method employing intrinsic protein fluorescence registration. In the temperature range from 10 to 30 degrees C the kinetic curves of Ca2+ and Mg2+ dissociation are best fitted with a sum of two exponential terms, each term is ascribed to a dissociation process in one of two bindings sites of parvalbumin. Dissociation rate constants in this temperature range increase from 0.03 to 0.8 s-1 and from 0.18 to 5 s-1 for Ca2+, and from 0.9 to 4.5 s-1 and from 4 to 33 s-1 for Mg2+. Parvalbumin equilibrium binding constants of Ca2+ and Mg2+ were also measured in the same temperature range. It makes possible to estimate the rate constants of association of Ca2+ and Mg2+. In the case of Ca2+ the rate of association approaches the diffusion controlled limit.

[Protein fluorescence of photosynthetic reaction centers from Rhodopseudomonas sphaeroides]. / O belkovoi fluorestsentsii fotosinteticheskikh reaktsionnykh tsentrov iz Rhodopseudomonas sphaeroides.

Luminescence emitted by tryptophan residues of reaction center (RC) preparations was studied. The RG preparations were isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides by treatment with lauryl dimethyl amine oxide (LDAO). After excitation at lambda 280 nm the quantum yield of luminescence is 0,02. It is shown that 60% of tryptophanyls are located inside the protein globule in the surrounding of relaxating polar groups and the rest approximately 40% on the outer surface of the globule--predominantly in the positively charged region of the LDAO-RC protein--in the surrounding of protein-bound water molecules. There is a correlation between the pH dependencies of the position of the peak of luminescence from tryptophanyls and effectivity of electron transfer from the primary (quinone) to secondary acceptor. The two parameters are invariant at pH from 7 to 9 and vary at pH less than 7 and pH greater than 9. The phenomena responsible for the observed correlation are discussed on the basis of pH-dependent changes in the RC protein which govern electron transport activity at the reaction center.