Influence of a Single Point Mutation in the Constant Domain of the Bence-Jones Protein bif on Its Aggregation Properties.

Multiple myeloma nephropathy occurs due to the aggregate formation by monoclonal immunoglobulin light chains (Bence-Jones proteins) in kidneys of patients with multiple myeloma. The mechanism of amyloid deposit formation is still unclear. Earlier, the key role in the fibril formation has been assigned to the variable domains that acquired amyloidogenic properties as a result of somatic mutations. However, fibril formation by the Bence-Jones protein BIF was found to be the function of its constant domain. The substitution of Ser177 by Asn in the constant domain of the BIF protein is most likely an inherited than a somatic mutation. To study the role of this mutation in amyloidogenesis, the recombinant Bence-Jones protein BIF and its mutant with the N177S substitution typical for the known immunoglobulin Cκ allotypes Km1, Km1,2, and Km3 were isolated. The morphology of aggregates formed by the recombinant proteins under conditions similar to those occurring during the protein transport in bloodstream and its filtration into the renal glomerulus, in the distal tubules, and in the proximal renal tubules was analyzed by atomic force microscopy. The nature of the aggregates formed by BIF and its N177S mutant during incubation for 14 days at 37°C strongly differed and depended on both pH and the presence of a reducing agent. BIF formed fibrils at pH 7.2, 6.5, and 10.1, while the N177S mutant formed fibrils only at alkaline pH 10.1. The refolding of both proteins in the presence of 5 mM dithiothreitol resulted in the formation of branched structures.

[Conformation of the ribosomal protein S1 of Thermus thermophilus in solution under different ionic conditions].

The structure of protein SI of Thermus thermophilus (M = 61 kDa) in solution at low and moderate ionic strengths (0 M and 100 mM NaCl, respectively) has been studied by small-angle X-ray and neutron scattering. It was found that protein S1 has a globular conformation under both ionic conditions. The modelling of different packing of six homologous domains of S1 on the basis of the NMR-resolved structure of one domain showed that the best fit of calculated scattering patterns from such complexes to experimental ones is observed at a compact package of the domains. The calculated value of the radius of gyration of the models is 28-29 angtroms, which is characteristic for globular proteins with a molecular mass of about 60 kDa. It was found that protein S1 has a tendency to form associates, and the type of the associate depends on ionic strength. These associates have, in general, two or three monomers at a moderate ionic strength, while at a low ionic strength the number of monomers exceeds three and they are packed in a compact manner. Strongly elongated associates were observed in neutron experiments at a moderate ionic strength in heavy water. The association of protein molecules was also confirmed by the data of dynamic light scattering. From these data, the translational diffusion coefficient of protein S1 at a moderate ionic strength was calculated to be (D20,w = (2.7 +/- 0.1) x 10(-7)cm2/s). This value is essentially smaller than the expected value (D20,w = (5.8 - 6.0) x 10(-7)cm2/s) for the S1 monomer in the globular conformation, indicating the association of protein molecules under equilibrium conditions.

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.

GroES co-chaperonin small-angle X-ray scattering study shows ring orifice increase in solution.

GroES consists of seven identical 10 kDa subunits and is involved in assisting protein folding as the partner of another oligomeric protein, the GroEL chaperonin. Here we studied the GroES structure in solution using small-angle X-ray scattering (SAXS). The SAXS pattern, calculated for the GroES crystal structure, was found to be different from the experimental one measured in solution. The synchronic shift in the radial direction and some turning of the protein subunits eliminate the difference and result in the increase of the hole diameter in the GroES ring-like structure from 8 A in the crystal to 21 A in solution.

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.

[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.

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.

[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.

[The functional value of complete dentures depending on the arrangement of the artificial teeth]. / Funktsional'naia tsennost' polnykh protezov v zavisimosti ot postanovki iskusstvennykh zubov.

Adhesion, functional adhesion, and observation of mechanical regularities during repair of dentition in patients with complete loss of teeth ensure stability of complete dentures and, hence, their functional value. Since functional adhesion is frequently impossible to provide as far as it concerns the lower denture, such dentures are always liable to the action of dumping force. The authors describe a method for making artificial teeth of complete removable dentures with due consideration for the individual force of occlusion of the jaws and the teeth inclination angle determined individually for each patient.

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.

[Characteristics of the electrical values of metal denture parts in patients wearing plate dentures]. / Kharakteristika élektricheskikh velichin mezhdu metallicheskimi chastiami zubnykh protezov u bol'nykh, pol'zuiushchikhsia plastinochnymi protezami.

The electric characteristics were lowered or absent in 87 patients wearing removable metal-based dentures. The explanation is as follows: in patients with plastic plate dentures and permanent metal prostheses in the oral cavity the electric charges pass between the prostheses via the saliva and oral tissues; in patients with metal-based plate dentures the charges pass along the metal basis, which fact is due to high electric conductivity of metal. Therefore in patients with plastic removable dentures the electric charges have a more detrimental effect on the oral tissues.

[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.

Correlation between enzyme activity and hinge-bending domain displacement in 3-phosphoglycerate kinase.

Diffuse X-ray-scattering data give evidence for large-scale structural change in pig muscle 3-phosphoglycerate kinase upon substrate binding. Simultaneous binding of 3-phosphoglycerate and MgATP either to the unmodified enzyme or to its active methylated derivative leads to about an 0.1-nm decrease in radius of gyration. These data coincide well with the previous data for yeast 3-phosphoglycerate kinase. When, instead of methylation, the two reactive thiol groups of pig muscle 3-phosphoglycerate kinase are carboxamidomethylated, the enzyme becomes inactive and the radii of gyration of its 'apo' and 'holo' forms do not differ within limits of experimental error. Thus, a correlation exists between the activity of 3-phosphoglycerate kinase and its substrate-induced large-scale conformational change. This correlation is a strong argument in favor of the functional importance of domain locking in the reaction catalyzed by 3-phosphoglycerate kinase.

[Large-scale structural changes of yeast phosphoglycerate kinase molecule upon substrate binding]. / Krupnomasshtabnye strukturnye izmeneniia v drozhzhevoi fosfoglitseratkinaze pri sviazyvanii substratov.

Changes of dimensions and form of the yeast phosphoglyceratekinase molecule upon ATP and 3-phosphoglycerate binding have been investigated by diffuse X-ray scattering. It has been shown that simultaneous sorption of both substrates is accompanied by an essential decrease both of the radius of gyration and the extent of asymmetry of the phosphoglyceratekinase molecule.

[Wide-angle x-ray scattering comparison of the structure of crystalline cytochrome c and cytochrome c in solution]. / Sravnenie struktury tsi tokhroma c v kristalle i rastvore metodom rasseianiia rentgenovskikh luchei pod bol'shimi uglama.

Large-angle X-ray diffuse scattering has been used for studying the conformational changes in cytochrome c during its transition from crystal into solution and during a change of the electron state of the heme. It has been found that the structure of cytochrome c in solution differs from its structure in crystal by a shift of the chain fragment in the region of 60-77 amino acid residues. The studies of the oxidized, reduced and cyanoforms of protein in solution have not revealed noticeable changes in the protein structure.