During their long existence, crustaceans, despite the absence of a highly specific adaptive immune system, like vertebrates, have successfully adapted to survive in their natural habitat that is rich in microorganisms, in particular, due to antimicrobial peptides. One valuable source of antimicrobial peptides is the hepatopancreas (HPC), which is a waste product of crab fishing and processing. Using the method of zymography and 1H-NMR spectroscopy we found that an extract from the hepatopancreas of the snow crab contains a small peptide (about 3 kDa) that hydrolyzes the cell wall and polysaccharide of the cell wall of M. lysodeikticus. This peptide may be of interest for practical use. A protein (about 14 kDa) was isolated from the hepatopancreas of the Red king crab using heparin–sepharose chromatography, which also exhibits activity against the cell wall of the gram-positive bacterium M. lysodeikticus, as was shown by zymography and turbidimetry.

The effectiveness of 1H-NMR-based metabolomic analysis for the detection of metabolic changes during carcinogenesis was assessed based on a comparison of the quantitative composition of metabolites in the blood plasma of healthy rats and rats that receiving M-1 sarcoma grafts. Plasma was collected from the rats under study on day 12 and day 36 after sarcoma transplantation to identify metabolites associated with tumor development. Analysis of the NMR spectra using multivariate statistical methods showed differences in the composition of metabolites for the groups of animals under study as soon as on day 12 after sarcoma transplantation; on day 36 the differences were significant. Twenty three metabolites were quantified. On day 12, only the lactate and allantoin levels were significantly different between the groups, while on day 36, the levels of nine metabolites were different between the two groups. All of the identified metabolites are involved in cancer metabolism, which makes 1H-NMR spectroscopy a promising method for cancer diagnosis.

Abstract—Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are β-sandwiches with a predominant β-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction, and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphologies in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The “KCl-aggregates” according to AFM data had the form of a “sponge”-like structure, while amorphous “glycine-aggregates” of titin formed “branching” structures. Spectrofluorometry revealed the ability of “glycine-aggregates” of titin to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross β-structure, a reflection of 4.6 Å, in these aggregates. These data indicate that “glycine-aggregates” of titin are amyloid or amyloid-like. No similar structural features were found in “KCl-aggregates” of titin; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data we obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.

The process of amyloid aggregation is quite complex and poorly studied. In this paper, summarizing the previously obtained results on the aggregation of the multidomain smooth muscle protein titin, we tried to complement the idea of its amyloid aggregation by presenting a new, in our opinion, possible mechanism. The main conclusion is that the ability of titin to form amorphous aggregates seems to be the only possible means of aggregation of this protein. Apparently, only individual sections of the molecules, and not the entire protein, are involved in the formation of the amyloid structure in amorphous aggregates of smooth muscle titin. This feature distinguishes titin from other amyloid or amyloid-like proteins due to the large size of the molecule. The possible energy landscape underlying the formation of amyloid aggregates of titin is discussed.

Protein oligomers are important intermediates in the formation of amyloid fibrils. In amyloidosis, for example, Alzheimer’s disease, oligomers can have a toxic effect on cells. This paper describes the distinctive features of oligomerization of multidomain muscle proteins, smooth muscle titin, and myosin-binding protein C (C-protein) of skeletal muscles consisting of FnIII-like and IgC2-like domains and capable of forming amyloid amorphous aggregates in vitro. Under conditions of low ionic strength (below physiological values), the C-protein formed stable oligomers that were not involved in further aggregation. Smooth muscle titin formed oligomers under conditions of high ionic strength (μ 0.6), which were precursors of amyloid amorphous aggregates of this protein.The results we obtained expand the understanding of the process of protein aggregation.

The process of amyloid aggregation is quite complex and poorly understood. In this work, having summarized previously obtained results on the aggregation of the multidomain smooth muscle protein titin, an attempt has been made to expand understanding of this process, and a new possible mechanism by which amyloid aggregation of titin may occur is delineated. Our main conclusion is that the ability of titin to form amorphous aggregates seems to be the only possible way of aggregation of this protein. Most likely, only separate parts of the molecules, but not the whole protein, are involved in the formation of the amyloid structure in amorphous aggregates of smooth muscle titin. This feature, given the large size of the protein molecule, distinguishes titin from other amyloid or amyloid-like proteins. The paper discusses the potential energy landscape underlying the formation of titin amyloid aggregates.

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.

Samples of rat homogenated brain cortex and cerebrospinal fluid prepared by various protocols were investigated by high-resolution NMR. The stability of the major metabolites, biomarkers of neurode-generative diseases, was examined depending on the procedure of sample pretreatment. As shown, the optimal conditions of the sample pretreatment of brain tissues to NMR analysis involve mechanical homogenization followed by the addition of deuterated water and for prolong experiments, an extraction with a methanol-chloroform mixture (2: 1). Samples of cerebrospinal fluid for NMR study should be prepared in buffer media based on deuterated water to improve the stability of the preparation and prevent the drift of metabolite signals in the spectra of different samples during the experimentation. The chosen optimal conditions of sample pretreatment of brain tissue and cerebrospinal fluid can be used in future to study changes in the composition and concentration of metabolites in neurodegenerative diseases by virtue of NMR. The proposed methods allow more accurate the diagnosis of pathologies of the brain function at early stages.

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.

Protein SHA-D of the SH3-Bergerac chimeric proteins family was constructed by the substitution of the β-turn N47-D48 in the spectrin SH3 domain by the KATANDKTYE amino acid sequence. The structural and dynamic properties of SHA-D in the solution were studied by means of high-resolution NMR spectroscopy. The extension of the SHA-D polypeptide chain in comparison with the wild type of protein WT-SH3 (∼17%) almost does not affect the overall molecule topology. The spatial structure of SHA-D is nearly identical to those of the proteins of the SH3-Bergerac family; however, there are some differences in the dynamic characteristics in the region of the insertion. The G52D substitution in the SHA-D protein results in the destabilization of the insertion region, where the conditions for the conformational exchange appear. The destabilization further affects the entire SHA-D molecule, making its structure more labile.

In order to further elucidate structural and dynamic principles of protein self-organization and protein-ligand interactions, a new chimeric protein was designed and a genetically engineered construct was created. 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 dynamic properties of the protein were studied with high-resolution NMR. According to NMR data, the tertiary structure of the chimeric protein SH3-F2 has a topology that is typical for 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 interactions of the ligand and the part of globule related to SH3 domain is not too large, because the analysis of protein dynamical characteristics points to the low amplitude, high-frequency ligand tumbling relative to the slow intramolecular motions of the main globule. The constructed chimera allows carrying out further structural and thermodynamic investigations of polyproline helix properties and its interaction with regulatory domains.

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 β-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 β-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.

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.

Covalent binding of a synthetic DNA fragment with eukaryotic transcription factor NF-κB 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-κB recognition site (κB-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-κB 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-α promoted partial translocation of the DNA reagent into the nucleus.

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-uracil glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence of amino acid residues, including a block of six His residues. In contrast to minimal differences in the amino acid sequences and functional activity, conformations of native and 6His-uracil glycosylases in solution were found to differ substantially at moderate ionic strength (60 mM NaCl). The structure of uracil-DNA glycosylase in solution is close to that in crystal and shows a tendency toward association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes partial dissociation of associates and compaction of protein structure. At the same time, 6His-uracil DNA glycosylase has a compact structure, intrinsically different from that in crystals. A decrease in the ionic strength of solution results in a partial destruction of the compact structure of the modified protein, keeping its functional activity unchanged.