[Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy].

Nicking endonuclease Nt.BspD6I is a heterodimeric restriction endonuclease, one subunit of which exhibits specific nicking activity. It gets bound to double-stranded DNA and makes a break (nick) in one chain at a distance of 4 nucleotides from the binding site. In this work, for visualization of the specific binding and protein landing site an atomic force microscopy was used. In five minutes after incubation of DNA solution with nicking endonuclease, the DNA molecules with associated proteins which located at the expected binding site and "shared" DNA strand into two segments (approximately, 1/3 and 2/3 of length) were observed in the images. In addition, near the binding site DNA molecule had a height corresponding to a single-stranded DNA molecule, which was in good agreement with single-stranded cleavage by nickase in the course of complex formation.

[Biophysics of single molecules].

The modern methods of research of biological molecules whose application led to the development of a new field of science, biophysics of single molecules, are reviewed. The measurement of the characteristics of single molecules enables one to reveal their individual features, and it is just for this reason that much more information can be obtained from one molecule than from the entire ensample of molecules. The high sensitivity of the methods considered in detail makes it possible to come close to the solution of the basic problem of practical importance, namely, the determination of the nucleotide sequence of a single DNA molecule.

[Family of ribosomal proteins S1 contains unique conservative domain].

Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.

[Physical methods and molecular biology].

The review is devoted to the description of the current state of physical and chemical methods used for studying the structural and functional bases of living processes. Special attention is focused on the physical methods that have opened a new page in the research of the structure of biological macromolecules. They include primarily the methods of detecting and manipulating single molecules using optical and magnetic traps. New physical methods, such as two-dimensional infrared spectroscopy, fluorescence correlation spectroscopy and magnetic resonance microscopy are also analyzed briefly in the review. The path that physics and biology have passed for the latest 55 years shows that there is no single method providing all necessary information on macromolecules and their interactions. Each method provides its space-time view of the system. All physical methods are complementary. It is just complementarity that is the fundamental idea justifying the existence in practice of all physical methods, whose description is the aim of the review.

[Prediction of short loops in the proteins with internal disorder].

New possibility of the FoldUnfold program for prediction of short disordered regions (loops), which appears by using the short window width (3 amino acid residues), was described. For three representatives of the proteins G family the FoldUnfold program predicted almost all short loops and yield results are well compatible with the X-ray structure data. We have classified the loops predicted in the protein Ras-p21 structure in two types. In the first type, loops have high values of the Debye-Waller factor typical of the so-called functional loops (flexible loops). In the other type, loops have lower values of the Debye-Waller factor and can be considered as loops connecting secondary structure elements (rigid loops). When the results of prediction with the use of our program are compared with the results of other programs (PONDR, RONN, DisEMBL, PreLINK, IUPred, GlobPlot 2, FoldIndex), it is seen that the first enables far better prediction of short loop positions. Use of FoldUnfold for ubiquitin-like domain h-PLIC-2 allows to resolve such task as definition of boundary between the structured and unstructured regions in proteins with a big portion of disordered regions. The FoldUnfold program defines a clear boundary between the structured and unstructured regions at amino acid residues 30-31,whereas each of the other programs outlines the boundary from the 28-th amino acid residues through the 70th.

[Structured proteins and proteins with internal disorder].

The point of view that a uniquely folded protein tertiary structure is required for the protein functioning has been prevailing in the literature quite recently. However of lately it has been found that many proteins in a cell have no such structure in an isolated state, though they have a well-defined function in physiological conditions. These proteins were named as proteins with natural or internal disorder. The portion of disordered regions in such proteins may vary from a sequence of several amino acids to a completely disordered sequence containing from tens to hundreds of amino acids. The main difference of these proteins from the structured (globular) ones is that they have no unique tertiary structure in an isolated state and acquire it after interaction with their partners. Their conformation in such a complex depends on the interacting partner and not only on their own amino acid sequence, which is specific for structured (globular) proteins. The problem of structural and functional relations in the structured proteins and proteins with internal disorder is discussed in this review. The complexity of the problem and its potential solutions are illustrated by the example of elongation factors EFlA.

[Unstructured regions in elongation factors EF1A from three overkingdom of the living world].

A new characteristic for classification of the Living World which based on the ability of amino acid sequences to form unstructured regions that appear as loops in their 3D structure is described. Our approach is in principle different from RNA and protein phylogenies that are based on the alignment of amino acid sequences from different organisms. Introduction of new structural-functional characteristic in itself is of undoubted interest because megataxonomy and macrophylogeny lack features that may be resolve evolutionary relation between different groups of organisms though apparent abundance of such characteristics are present. We used the program FoldUnfold to search for unstructured regions in the elongation factors EF1A. The reliability of loop prediction was checked against five factors whose structure is known from X-ray analysis. In addition to two cross-bridges between three structural domains in the elongation factors, the program predicts extra loops. Not counting the effector loop that is inherent to all factors, there are six. Three (A, B and C) of the six different loops are revealed in the first domain, one loop (D) in the second, and two loops (E and F) in the third domain of the factor, all six of which are never found in the same factor. Signatures of elongation factors for each Superkingdom of the Living World have been found for several dozen typical representatives from each Superkingdom. These signatures lead to the variation of the number of loops and their localization within the factor domains. The obtained data leads us to believe that the approach based on the prediction of unstructured protein loops--up to six--must have higher resolution than the method based on the indels (insertion + deletion), the number of which equals one for the same elongation factors. In our analysis, the specificity of sequences it is important, in addition to the existence of loops. Since the total number of loops predicted in the factors increases with the complexity of an organism, we propose the following about the role of the loops in evolution: holding to the principle of "thrifty inventiveness", Nature operates with different universal inserts (loops) adapting their number and location among the factor domains as well as their amino acid composition so that the protein will perform special functions: one in protozoa and several in higher organisms.

[Roughness of the globular protein surface]. / O prirode sherokhovatosti poverkhnosti belkovykh globul.

The area and volume of the approximating ellipsoids taken from low resolution X-ray data have been calculated for 65 globular proteins. It has been shown that the dependence of these values on the protein molecular mass (M) coincides with those for even isometric bodies. This indicates that the asymmetry of globular proteins does not grow with the increase of their sizes. At the same time the 0.73 slope of the log-log dependence of the accessible surface area (A(s)) on the protein molecular mass differing from the value of 0.67 for even isometric bodies was observed (Miller S. et al., J. Mol. Biol. 1987. V.196. P.641). This can be explained by peculiarities of the protein surface. The method of molecule shape recovery by spherical harmonics has shown that the domain organization of protein molecule cannot explain the observed difference. Therefore the more detailed analysis of protein surface structure would be necessary.

[Partially unfolded state of lysozyme with a developed secondary structure in dimethylsulfoxide]. / Chastichno razvernutoe sostoianie lizotsima s razvitoi vtorichnoi strukturoi v dimetilsul'fokside.

The conformation of a chicken egg lysozyme molecule (dimensions, stoichiometry of its associates, and the degree of helicity) in DMSO was studied by small-angle neutron scattering, dynamic light scattering, and optical rotatory dispersion in the visible region of the spectrum. At high DMSO concentrations (70%), the protein was shown to exist as a dimer. The monomer molecules in the dimer adopt a partially unfolded conformation, with dimensions substantially greater than those in the native state and a high content of secondary structure (the degree of helicity is close to that of native lysozyme). This approach provides a unique possibility to assess the compactness of molecules in associates, which may be very useful in studying protein self-organization.

[Changes in mechanical characteristics of immobilized crystals and films of globular proteins during substitution of D2O for H2O]. / Izmenenie mekhanicheskikh kharakteristik immobilizovannykh kristallov i plenok globuliarnykh belkov pri zamene H2O na D2O.

The influence of substitution of the isotopic composition of the medium on the mechanical properties of immobilized crystals and films from bovine pancreatic ribonuclease and hen egg white lysozyme was investigated. The order of magnitude of the observed effects indicates that the contribution of the electrostatic interaction to the observed isotopic effect may be considered inessential. The absence of aggregation in the H2O and D2O medium under experimental conditions is demonstrated by the method of the low angle dispersion of X-rays. The observed effects of D2O on the mechanical behavior of crystals and films of proteins may be accounted for by the strengthening of molecular interactions in the samples.

[Structure and density of ribosomal RNA and its complexes with proteins in a solution]. / O forme i kompaktnosti ribosomnykh RNK i ikh kompleksov s belkami v rastvore.

X-ray and neutron scattering, as well as velocity sedimentation, were used to study the shape and dimensions (compactness) of isolated ribosomal (16S and 23S) RNA's and their complexes with ribosomal proteins. The neutron scattering of ribosomal particles in 42% 2H2O where the protein component is contrast-matched, were taken as a standard of comparison characterizing the dimensions and shape of the 16S and 23S RNA in situ. This comparison allowed the following conclusions: (1) The shape of the isolated 16S RNA at a sufficient Mg2+ concentration (e. g., in the reconstruction buffer) is similar to that of the 16S RNA in situ, but its compactness is somewhat less. (2) The 16S RNA in the complex with protein S4 has a shape and compactness similar to those of the isolated 16S RNA. (3) The 16S RNA in the complex with four core proteins, namely S4, S7, S8 and S15, has a shape and compactness similar to those of the isolated 16S RNA. (4). The six ribosomal proteins, S4, S7, S8, S15, S16, and S17, are necessary and sufficient for the 16S RNA to acquire a compactness similar to that in situ.

[Isolation and physical study of the 13S fragment of 16S RNA and its complex with ribosomal protein S4]. / Vydelenie i fizicheskoe issledovanie 13S fragmenta 16S RNK i ego kompleksa s ribosomnym belkom S4.

A fragment of E. coli 16S RNA has been obtained by its hydrolysis with pancreatic RNAase A coupled to Sepharose 4B. This fragment has a molecular weight of 170 000 and a sedimentation coefficient of 13S. It does not aggregate in solution and binds with the ribosomal protein S4. The 13S fragment and it complex with the protein S4 have been studied by different physical methods in the first place, by neutron scattering. It has been shown that this fragment is compact in solution. The radii of gyration of the fragment (50 +/- 3 A) and of the protein S4 within the complex (17 +/- 3 A) coincide, within limits of experimental error, with the radii of gyration for the free RNA fragment (47 +/- 2 A) and the free ribosomal protein S4 in solution (18 +/- 2 A). Hence, the conclusion is made that the compactness of the 13S fragment of the 16S RNA and the ribosomal protein S4 does not change at the complex formation. The compact 13S fragment of the 16S RNA is shown to be contrast matched in the H2O/D2O mixture containing 70% D2O which corresponds to its partial specific volume v equal to 0.537 cm3/g.

[Quaternary structure of the ribosomal 30S subparticle: the model and its experimental verification]. / Chetvertichnaia struktura ribosomnoi 30S subchastitsy: model' i ee eksperimental'naia proverka.

In considering the structure of the ribosomal 30S subparticle from Escherichia coli we have assumed that : 1) all or almost all the proteins in the 30S subparticle are compact and globular as has been shown for isolated proteins S4, S7, S8, S15 and S16 in solution [Serdyuk I. N., Zaccai G. and Spirin A. S. (1978) FEBS Letters, 94, 349-352]; 2) RNA within the 30S subparticle has the same specific V-like or Y-like shape demonstrated for the isolated 16S RNA in a compact conformation [Vasiliev V. D., Selivanova O. M. and Koteliansky V. E. (1978) FEBS Letters, 95, 273-276]. On the basis of this assumption and numerous data published on the mutual localization of ribosomal proteins, we have constructed a model of the quaternary structure of the ribosomal 30S subparticle. We have tested the model by comparing the theoretically calculated curves of neutron and X-ray scattering at different contrasts with the corresponding experimental scattering curves of the E. coli 30S subparticles and have found that they coincide. The calculated scattering curves of several previously published three-dimensional diagrams of protein topography in the 30S subparticle do not agree with experiment.

[Internal structure of ribosomes using different types of emission]. / Izuchenie vnutrennei struktury ribosom s pomoshch'iu razlichnykh tipov izlucheniia.

A review is made of the experimental results obtained by the author and co-workers on the study of the structural organization of the RNA and the protein in ribosomes by the method of joint use of light, X-ray and neutron scattering and by the method of contrast variation in neutron scattering. Two rules are formulated for the folding of the ribonucleoprotein strand in ribosomes: (1) in each ribosomal subparticle the RNA is concentrated predominantly closer to the center of the particle whereas the protein has a more peripherical localization; (2) the compact ("crystallic") packing of hydrated RNA helices is an essential feature of the nucleus (nuclei) organization of the particles. An analysis of the experimental data on neutron scattering by ribosomal proteins has been done and the globulin conformation in solution of some of these proteins has been established. The widespread concept according to which the majority of ribosomal proteins on the ribosome and in solution are enlongated expanded structures is disputed. It is suggested that all, or almost all, ribosomal proteins are usual globular proteins recognizing the specific sequence of RNA on the periphery of the particles, and , hence, that the formation of functional centrers on the ribosome is, in principle, analogous to the formation of functional centers of other complex proteins with a quaternary structure.