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.

Destabilization of CH2 domains in intact IgG2 is accompanied by reduced ability to inhibit complement system factor C1.

Fc fragments (hFc) of human myeloma IgG2 proteins LOM and SIN having core hinge (Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys) were first obtained by a modified proteolytic procedure. The thermostability of CH2 domains inside of standard Fc, hFc fragments, and intact IgG2 LOM and SIN was studied by fluorescence spectroscopy. It was found that CH2 domains of intact IgG2 are destabilized. The destabilization is accompanied by reduced ability of IgG2 to inhibit the activation of complement system by classical pathway. This could be due to the decrease in the affinity of CH2 domains to factor C1q.

Design of specific gratings operating under surface plasmon-polariton resonance.

We present a productive method of designing specific diffraction gratings that ensure some predetermined energy redistributions between outgoing waves. It is based on the analytical solution of the direct and reverse diffraction problems under surface plasmon-polariton resonance and is supported by strict numerical calculations.

[Chimeric SHA-D domain ("SH3-Bergerac"): 3D-structure and dynamics studies in solution].

Protein SHA-D of "SH3-Bergerac" chimeric proteins family was constructed by substitution of beta-turn N47-D48 in spectrin SH3-domain by KATANDKTYE amino acid sequence. Structural and dynamics properties of SHA-D in solution were studied by with the help of high-resolution NMR. The extension of SHA-D polypeptide chain in comparison with wild type of protein WT-SH3 (~ 17%) practically doesn't affect almost the total molecule topology. 3D-structure of SHA-D is practically identical to the proteins of "SH3-Bergerac" family. However there are some differences in dynamic characteristics in the region of substitution. The G52D substitution in SHA-D protein results in a destabilization of the region insertion where the conditions for conformational exchange appear. Destabilization further affects the entire SHA- D molecule making its structure more labile.

[NMR structure and dynamics of the chimeric protein SH3-F2].

For the further elucidation of structural and dynamic principles of protein self-organization and protein-ligand interactions the design of new chimeric protein SH3-F2 was made and genetically engineered construct was created. The SH3-F2 amino acid sequence consists of polyproline ligand mgAPPLPPYSA, GG linker and the sequence of spectrin SH3 domain circular permutant S19-P20s. Structural and dynamics properties of the protein were studied by high-resolution NMR. According to NMR data the tertiary structure of the chimeric protein SH3-F2 has the topology which is typical of SH3 domains in the complex with the ligand, forming polyproline type II helix, located in the conservative region of binding in the orientation II. The polyproline ligand closely adjoins with the protein globule and is stabilized by hydrophobic interactions. However the interaction of ligand and the part of globule relative to SH3 domain is not too large because the analysis of protein dynamic characteristics points to the low amplitude, high-frequency ligand tumbling in relation to the slow intramolecular motions of the main globule. The constructed chimera permits to carry out further structural and thermodynamic investigations of polyproline helix properties and its interaction with regulatory domains.

[Study of the structure and dynamics of a chimeric variant of the SH3 domain (SHA-Bergerac) by NMR spectroscopy].

A structural-dynamic study of one of the chimeric proteins (SHA) belonging to the SH3-Bergerac family and containing the KATANGKTYE sequence instead of the N47D48 beta-turn in the spectrin SH3 domain was carried out by high resolution NMR spectroscopy. The spatial structure of the protein was determined and its dynamics in solution was investigated on the basis of the NMR data. The elongation of the SHA polypeptide chain in comparison with the WT-SH3 original protein (by ~17%) exerts practically no effect on the general topology of the molecule. The presence of a stable beta-hairpin in the region of insertion was confirmed. This hairpin was shown to have a higher mobility in comparison with other regions of the protein.

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.

[Structure of Escherichia coli uracil DNA glycosylase and its complexes with nonhydrolyzable substrate analogues in solution observed by synchrotron small-angle X-ray scattering].

The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.

[DNA duplexes containing 2'-deoxy-2'-iodoacetamidouridine as reagents for affinity modification of proteins]. / DNK-dupleksy, soderzhashchie 2'-dezoksi-2'-iodatsetamidouridin, kak reagenty dlia affinnoi modifikatsii belkov.

DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.

Transition-metal complexes as inhibitors of proteins recognizing double-stranded fragments of nucleic acids.

A 30-membered DNA duplex containing the recognition site of restriction endonuclease SsoII and a 30-membered RNA--DNA hybrid duplex, a substrate of E. coli RNase H, were synthesized. The cleavage of the 30-membered fragments of nucleic acids catalyzed by endonucleases in the presence of Co-phthalocyanine complex [CoPc(COONa)8] containing eight carboxyl groups at the periphery of the ligand was studied. It was shown that the efficiency of enzyme catalysis decreases in the presence of the metal complex for both endonucleases. By addition of a 100-fold excess of Co-phthalocyanine complex with respect to DNA duplex the initial rate of substrate hydrolysis by restriction endonuclease SsoII is observed to decrease twice. An equimolar ratio of the metal complex and hybrid duplex leads to essentially complete inhibition of RNA cleavage by RNase H from E. coli. The inhibition of catalytic activity of enzymes recognizing the double-stranded nucleic acids in the presence of Co-phthalocyanine complex is assumed to be caused by the ability of the latter to interact with DNA, RNA, and DNA--RNA duplexes.