[Native globular and native partially or completely disordered proteins. Folding, supramolecular complex formation and aggregation].

Recently it became evident that proteins can perform their function not only in globular state but also in partially or completely disordered state. The majority of globular proteins are enzymes which function is strictly determined. Regulation and signaling proteins participating in interconnection with variety of partners must have much more lability, and macromolecules of such proteins are mainly in partially or completely disordered state. The aim of this work was to describe from the unified viewpoint in the frame of energy landscape model the existence of native globular, native partially or completely disordered proteins, formation of intermolecular complexes with various partners, formation of amorphous aggregates and amyloid fibrils. Compact globular proteins are formed if polypeptide chain provides strong intramolecular interconnections. The ability of polypeptide chain to fold in a compact globule depends on the relation of hydrophobic and charged aminoacids in its composition. Many partially or completely disordered proteins can form compact structure in complexes with their partners, which are composed by intermolecular interactions of polypeptide chains of protein and its partner. Intermolecular interaction of proteins can lead to formation associates, amorphous aggregates, amyloid and amyloid-like fibrils. The requisite condition of such contact formation is the availability of hydrophobic clusters of polypeptide chain exposed to the solution. That is why aggregation of partially or completely disordered proteins is more favorable in comparison with globular proteins.

[The role of quarternary structure in fluorescent protein stability].

The stability of fluorescent proteins (FPs) is of great importance for their use as reporters in studies of gene expression, protein dynamics and localization in cell. A comparative analysis of conformational stability of fluorescent proteins, having different association state was done. The list of studied proteins includes EGFP (monomer of green fluorescent protein, GFP), zFP506 (tetramer GFP), mRFP1 and "dimer2" (monomer and dimmer of red fluorescent protein), DsRed1 (red tetramer). The character of fluorescence intensity changes induced by guanidine hydrochloride (GdnHCl) of these proteins differs significantly. Green tetramer zFP506 has been shown to be more stable than green monomer EGFP, red dimmer "dimer2" has been shown to be less stable than red tetramer DsRed1, while red monomer mRFP1 has been shown to be practically as stable as tetramer DsRedl. It is concluded that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stability.

[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.

[A new scheme for the isolation and purification of human alpha-fetoprotein]. / Novaia skhema vydeleniia i ochistki alpha-fetoproteina cheloveka.

A new efficient scheme for the isolation and purification of human alpha-fetoprotein from human cord serum was proposed. Sequential chromatography on four columns (Cibacron Blue Sepharose, two metal chelate, and one reversed-phase) helped rapidly prepare alpha-fetoprotein samples of high purity (no less than 98%; purification factor approximately 10(3)) in high yields (approximately 85%).

[Effect of hyperphosphorylation on structure of TAU protein]. / Vliianie izbytochnogo fosforilirovaniia na strukturnye svoistva belka tau.

Effect of hyperphosphorylation on structural properties and conformational stability of tau-protein was investigated by methods of circular dichroism and fluorescence decay. The normal protein displayed an unusual secondary structural elements--an extended left-handed helix. It is suggested that the structure of the normal tau-protein includes a globular C-terminal part with rigid extended tail, i.e., it is of a "tadpole" type. The normal protein structure is practically unaffected by changes in pH values. Hyperphosphorylation leads to some perturbation within the extended part of the protein molecule. Decrease in pH transforms the globular part of the hyperphosphorylated protein into the molten globule-like conformation.

[ANS fluorescence. I. Effect of self-association on the spectral properties of the probe]. / Fluorestsentsiia ANS. I. Vliianie samoassotsiatsii na spektral'nye svoistva zonda.

The dependence of spectral properties of Mg2+ and NH4+ salt of 8-anilino-1-naphthalenesulfonic acid (Mg-(ANS)2 and NH4-ANS, respectively) on the dye concentration and solvent composition was investigated by means of steady-state and time-resolved fluorescence spectroscopy. We have shown that the increase in ANS concentrations leads to changes in the shape of absorption and fluorescence spectra of the dye, accompanied by the decrease in its fluorescence decay time values. Such changes, observed in aqueous and organic solvents for both salts of ANS, reflect the existence of self-association of the dye molecules. The decrease in fluorescence intensity induced by self-association of the probe molecules is too small to explain a weak fluorescence of ANS in water. At the same time, it expounds the difference between the decay times of protein-embedded ANS molecules upon interaction of this probe with native and molten globule proteins.

[ANS fluorescence. II. Use of the method of fluorescent probe decay for studying the structural transformations in globular proteins]. / Fluorestsentsiia ANS. II. Primenenie metoda zatukhaniia fluorestsentsii zonda dlia issledovaniia strukturnykh prevrashchenii v globuliarnykh belkakh.

Changes in ANS fluorescence decay parameters induced by the interaction of the probe with proteins have been investigated. The existence of at least two different modes of interactions between the ANS and protein was established. The interactions of the first type are connected with binding of an ANS molecule with the surface of a protein molecule. In this case ANS molecules are well acceptable for a solvent. The interactions of the second type are characteristic of the protein-embedded ANS molecules. The decay time values of the second type complexes change considerably (> 1.5-fold) during the protein molecule transformation into the molten globule-like conformation. The molecular model explaining such a behaviour is suggested.

[Point amino acid substitutions in the Ca2+-binding centers of recoverin. I. Mechanism of successive filling of Ca2+-binding centers]. / Tochechnye aminokislotnye zameny v Ca2+-sviazyvaiushchikh tsentrakh rekoverina. I. Mekhanizm posledovatel'nogo zapolneniia Ca2+-sviazyvaiushchikh tsentrov.

The molecule of photoreceptor Ca(2+)-binding protein recoverin contains four potential Ca(2+)-binding sites of the EF-hand type, but only two of them (the second and the third) can actually bind calcium ions. We studied the interaction of Ca2+ with recoverin and its mutant forms containing point amino acid substitutions at the working Ca(2+)-binding sites by measuring the intrinsic protein fluorescence and found that the substitution of Gln for Glu residues chelating Ca2+ in one (the second or the third) or simultaneously in both (the second and the third) Ca(2+)-binding sites changes the affinity of the protein to Ca2+ ions in different ways. The Gln for Glu121 substitution in the third site and the simultaneous Gln substitutions in the second (for Glu85) and in the third (for Glu121) sites result in the complete loss of the capability of recoverin for a strong binding of Ca(2+)-ions. On the other hand, the Gln for Glu85 substitution only in the second site moderately affects its affinity to the cation. Hence, we assumed that recoverin successively binds Ca(2+)-ions: the second site is filled with the cation only after the third site has been filled. The binding constants for the third and the second Ca(2+)-binding sites of recoverin determined by spectrofluorimetric titration are 3.7 x 10(6) and 3.1 x 10(5) M-1, respectively.

[Effect of a biologically active interferon fragment on the structure of the synthetic protein carrier]. / Issledovanie vliianiia biologicheski aktivnogo fragmenta interferona na strukturu iskusstvennogo belka-nositelia.

A biologically active de novo protein albeferon was studied by physical techniques including CD spectroscopy in far and near ultraviolet regions and microcalorimetry. Albeferon was obtained by grafting an active octapeptide interferon fragment into a de novo protein albebetin used as a carrier. It was shown that attachment of the octapeptide into its molecule did not weaken albebetin but even slightly improved its structure and stability. The obtained results can be used for de novo protein design and improvement.

[How many molten globules states exist?]. / Skol'ko sushchestvuet razlichnykh rasplavlennykh globul?.

The problem of a variety of denatured forms of the protein molecule under equilibrium conditions is considered. The experimental conditions are described at which the protein molecule can exist in various non-native states. The history of the discovery of a universal intermediate molten globule state and the current status of research in this field are briefly outlined. Particular emphasis is placed on the fact that the molten globule state is a thermodynamic state of the protein molecule that is separated from both the native and the completely unfolded state by "all-or-none" transitions, i.e., intramolecular analogs of the 1st-order phase transitions. It is also shown that the molten globule state is not the only intermediate state observed for a particular protein under equilibrium conditions. The main structural features of the protein molecule in various denatured conformations are described. How many molten globule states there exist? A molten globule, a precursor of the molten globule, a highly structured molten globule: are these particular conformational states or different forms of the unique intermediate state? Or different forms of the native protein molecule with different degrees of disorder? Or differently structured forms of the unfolded polypeptide chain? This review is an attempt to answer these questions.

[Stabilization of the alpha-fetoprotein structure with sucrose]. / Stabilizatsiia struktury alpha-fetoproteina sakharozoi.

By means of scanning microcalorimetry and fluorescent spectroscopy, the addition of sucrose was shown to stabilize the structure of human alpha-fetoprotein (AFP). The stabilizing effect was not eliminated during eight-day dialysis of AFP against a buffer containing no sucrose, but it can be substantially weakened by treating AFP with a specific enzyme, invertase, which splits sucrose into fructose and glucose. This indicates that human AFP is capable of specific sucrose binding.

[Structural properties of ribosomal protein S8 from the extreme thermophile Thermus thermophilus]. / Strukturnye svoistva riboxomnogo belka S8 iz ékstremal'nogo termofila Thermus thermophilus.

The gene of ribosomal protein S8 from the extreme thermophile Thermus thermophilus was expressed in E. coli using the strain BL21(DE3) and vector pET3-1. A method of isolating this protein from the super producing strain was developed, which makes it possible to obtain 8-12 mg of product from 11 of culture. The secondary structure of protein S8 was determined by using CD spectroscopy. The protein was shown to be highly resistant to denaturants.