Loops and repeats in proteins as footprints of molecular evolution.

This review is devoted to substantiation of new characteristics for classification of living organisms. The novel view of a role of flexible regions in protein functioning and evolution is suggested. It is based on the newly revealed correlation between the number of loops in elongation factors and the complexity of organisms. This correlation allowed us to formulate a hypothesis of evolution of this protein family. In addition, the study of the ribosomal protein S1 family made it possible to consider the number of structural domains as a reliable indicator of a microorganism's affiliation with a particular division and to judge about "direction" of their evolution. The findings allow us to consider the loops and repeats in these proteins as unique imprints of molecular evolution.

YB-1 is capable of forming extended nanofibrils.

Here we are the first to report that multifunctional Y-box binding protein 1 (YB-1) forms extended fibrils with a diameter of 15-20 nm. The YB-1 fibrils were visualized by atomic force and electron microscopy after 1-h incubation in solution with 2 M LiCl. Their length grew with incubation time and could exceed 10 microm; their shape is helical or zigzag-like. They possess polarity and tend to associate with one another to give structures of a higher order, like ribbons or bundles. The YB-1 fibrillar architecture has a distinct periodicity with a repeat unit of about 52 nm.

Modified DNA fragments specifically and irreversibly bind transcription factor NF-kappaB in lysates of human tumor cells.

Covalent binding of a synthetic DNA fragment with eukaryotic transcription factor NF-kappaB has been studied in lysates of human colon carcinoma HCT-116 cells. For binding we used 32P-labeled 17/19 bp nucleotide DNA duplex containing an NF-kappaB recognition site (kappaB-site) in which one of internucleotide phosphate groups was replaced by a chemically active trisubstituted pyrophosphate group. Using gel electrophoresis under denaturing conditions (Laemmli electrophoresis) followed by immunoblotting revealed selective irreversible binding of 32P-labeled DNA duplex with NF-kappaB in lysates of tumor cells in the presence of other cell components. Experiment on delivery of this DNA duplex containing rhodamine at 3 -end of the modified chain in an intact cell revealed that rhodamine-labeled DNA penetrated through the plasma membrane of tumor cells without any additional delivery systems. Using fluorescent microscopy, we found that the rhodamine-labeled DNA is initially localized in the cytoplasm. Confocal laser scanning microscopy revealed that subsequent treatment of the cells with TNF-alpha promoted partial translocation of the DNA reagent into the nucleus.

Extended conformation of mammalian translation elongation factor 1A in solution.

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.

Roughness of the globular protein surface: analysis of high resolution X-ray data.

In an earlier publication [Serdyuk, I.N. et al., Biofizika, in press, 1997] we demonstrated that the asymmetry extent of globular proteins does not change with increasing their sizes, and the observed nontrivial dependence of the protein accessible surface area on the molecular mass [Miller, S., J. Mol. Biol. 196:641-656, 1987] (A(s) - M dependence) is a reflection of the protein surface relief peculiarities. To clarify these peculiarities, an analysis of the molecular surface on the basis of high-resolution x-ray data has been done for 25 globular proteins not containing prosthetic groups. The procedure was based on studying the dependence of the minimal number (N) of probe bodies (here cubes) covering the entire protein surface, both on their size (N - R dependence) and on the value of dry protein volume (N - V dependence). Two levels of protein surface organization have been detected by molecular surface analysis. On the micro scale (2-7 A), the surface is characterized by a D = 2.1 fractal dimension which is intrinsic to surfaces with weak deformations and reflects the local atomic group packing. On the macro scale, large-scale surface defects are revealed that are interpreted as the result of secondary structure elements packing. A simple model of protein surface representation reflecting large-scale irregularities has been proposed.

Preparation of a 'beheaded' derivative of the 30S ribosomal subunit.

Oligodesoxyribonucleotide-directed cleavage of protein-deficient Thermus thermophilus derivatives of the 30S ribosomal subunits with RNase H is described. A homogeneous RNP fragment has been isolated as a result of the cleavage and subsequent purification in the sucrose gradient. It corresponds to the central and 5' domains of the 30S ribosomal subunit. The high compactness of the fragment in solution suggests that it can be considered as a 'beheaded' derivative of the 30S ribosomal subunit. The absence of a reconstitution stage in isolation of the 22S RNP fragment provides for its preparation in large amounts.

Protein globularization during folding. A study by synchrotron small-angle X-ray scattering.

Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS). SAXS patterns of proteins and model polypeptides in globular states (native and "molten globule") and in non-globular states (unfolded protein as well as randomly coiled, partially alpha-helical and partially beta-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots. Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass. The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra. The intermolecular association of the protein "molten globule"-like intermediates accumulated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters. It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration. We propose the integrated SAXS intensity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions. Time-resolved refolding curves of the above proteins were monitored by the integrated SAXS intensity to investigate the globularization process in protein folding. Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded. The kinetic processes reflect both protein intramolecular globularization and its intermolecular association.

Structural model of the 50S subunit of E. coli ribosomes from solution scattering.

The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Based on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40 A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

The triple isotopic substitution method in small angle neutron scattering. Application to the study of the ternary complex EF-Tu.GTP.aminoacyl-tRNA.

The TIS (triple isotopic substitution) method in small angle neutron scattering was applied to determine the radius of gyration of polypeptide elongation factor Tu (EF-Tu) from E. coli associated with GDP and within the ternary complex EF-Tu.GTP.aminoacyl-tRNA. The results showed that, within errors of about 1 A, there is no change in the radius of gyration of the EF-Tu moiety upon ternary complex formation. Experiments were performed in H2O buffer, in which complex formation could be followed on an absolute scale because of the relatively large contrast of both protein and tRNA. The TIS method is based on the analysis of a scattering curve that is the difference between the scattering of two solutions containing appropriately deuterium labelled particles. A necessary condition for the application of the method is that the two solutions are identical in all respects except for the extent of deuterium label. The main properties of TIS that make it very useful for the study of complex particles in solution were confirmed by this study. These are the elimination of interparticle effects in the difference curve, the 'invisibility' of unlabelled parts of the particles and the independence of the difference scattering curve on the buffer 2H2O-H2O content. The last property is of particular interest for the study of interactions that may be influenced by 2H2O, since, contrary to classical contrast variation methods, TIS experiments can be performed in H2O buffer alone.

Solution scattering from 50S ribosomal subunit resolves inconsistency between electron microscopic models.

Models of the 50S ribosomal subunit from electron microscopy on isolated particles and on ordered sheets display significantly different features. A model of the shape of the native Escherichia coli 50S subunit in solution and of its RNA-rich core at 4-nm resolution has been produced by using methods for joint interpretation of x-ray and neutron small-angle scattering data obtained by contrast variation. The good agreement between the shape of the entire 50S subunit and the electron microscopic models of isolated particles and between the RNA-rich core and the model obtained from ordered sheets leads to the conclusion that the latter, which is based on the subjective contouring of density maps, is heavily biased toward the RNA.

Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering. I. X-ray synchrotron radiation study.

The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50 S ribosomal subunit of Escherichia coli in solution is described. The experimental X-ray data from contrast variation with sucrose are analysed in terms of the basic functions in real space and the scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins are obtained. From these curves models of the shape of the 50 S subunit and its RNA-rich core are evaluated. These two shapes are positioned so that their difference, which approximates the volume occupied by the proteins, produces a scattering curve which is in good agreement with the scattering from the protein moiety.

Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering. II. Neutron scattering study.

The X-ray and neutron contrast variation data of the 50 S ribosomal subunit of Escherichia coli in solution are interpreted in the frame of a two-phase model described by the shapes of the 50 S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50 S subunit and of the RNA-rich core are evaluated with a resolution of about 4 nm. The shape of the envelope is in good agreement with the models of the 50 S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

Structural changes in 16S RNA from Escherichia coli upon unfolding by urea.

The urea-induced unfolding of 16S RNA at low ionic strength has been studied by dynamic light scattering, uv spectroscopy, and some hydrodynamic methods. Three components could be resolved in the photon correlation spectra of scattered light, using the inverse Laplace transform SIPP program [G.R. Danovich and I.N. Serdyuk (1983) in Photon Correlation Techniques in Fluid Mechanics, vol. B38, E.O. Schulz-Dubois, Ed., Springer, Berlin/Heidelberg, New York, p. 315]. One component is assigned to the center-of-mass translation of the RNA, another one to a combination of translational and internal motion, and the last to diffusion of urea clusters. The hydrodynamic dimensions of RNA increase strongly upon transition from 4 to 6 M urea. We conclude that up to 2 M urea, 16S RNA is highly elongated, and coiled above 4 M urea, with a great increase of the hydrodynamic dimensions of RNA being observed upon transition from 4 to 6 M urea. A scheme for RNA unfolding is proposed.

Translocation makes the ribosome less compact.

Translating ribosomes of Escherichia coli were prepared either in the pre-translocation or in the post-translocation states by a special technique based on the use of poly(U)-Sepharose columns where the template was coupled to the matrix through splittable -S-S- bridges. Elongation factors were absent from the final preparations. A neutron scattering study of the translating ribosomes in the two functional states was performed at different contrasts (various 1H2O/2H2O mixtures). Under conditions of a high contrast for the protein constituent the radius of gyration of the post-translocation-state ribosomes was found to be slightly greater than that of the pre-translocation-state ribosomes. Using the results of this study the conclusion can be drawn that translocation is accompanied by a spatial displacement of some parts of the ribosome with a magnitude of several ångström units.

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering.

The structure of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500 +/- 4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume Vc, surface Oc, radius of gyration Rs, maximum dimension of the molecule L, thickness D, and cross section radius of gyration Rsq, agree within the experimental error limits. The mean values are Vc = 57 +/- 3 nm3, Oc = 165 +/- 10 nm2, Rs = 3.37 +/- 0.05 nm, L = 10.8 +/- 0.7 nm, D = 1.57 +/- 0.07 nm, Rsq = 0.92 +/- 0.01 nm. Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution functions. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D020, w = (5.9 +/- 0.2) X 10(-7) cm2 s-1 and (6.2 +/- 0.2) X 10(-7) cm2 s-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Shape and compactness of the isolated ribosomal 16 S RNA and its complexes with ribosomal proteins.

X-ray scattering, neutron scattering and velocity sedimentation techniques were used for studies of ribosomal 16 S RNA in the isolated state and in different complexes with ribosomal proteins. The neutron scattering curve of the ribosomal 30 S subparticle in 42% 2H2O where the protein component is contrast-matched, was taken as a standard of comparison characterizing the dimensions and shape of the 16 S RNA in situ. The following deductions result from the comparisons. The shape of the isolated 16 S RNA at a sufficient Mg2+ concentration (e.g., in the reconstruction buffer) is similar to that of the 16 S RNA in situ, i.e. in the 30 S particle, but it is somewhat less compact. The 16 S RNA in the complex with protein S4 has a shape and compactness similar to those of the isolated 16 S RNA. The 16 S 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 16 S RNA. The six ribosomal proteins S4, S7, S8, S15, S16 and S17 are necessary and sufficient for the 16 S RNA to acquire a compactness similar to that within the 30 S particle. The general conclusion is that the overall specific folding of the 16 S RNA is governed and maintained by its own intramolecular interactions, but the additional folding-up (about one-fourth of the linear size of the whole molecule) or the stabilization of the final compactness requires some ribosomal proteins.