Influence of Chaperones on Amyloid Formation of Ðß Peptide.

BACKGROUND: An extensive study of the folding and stability of proteins and their complexes has revealed a number of problems and questions that need to be answered. One of them is the effect of chaperones on the process of fibrillation of various proteins and peptides. METHODS: We studied the effect of molecular chaperones, such as GroEL and α-crystallin, on the fibrillogenesis of the Aß(1-42) peptide using electron microscopy and surface plasmon resonance. RESULTS: Recombinant GroEL and Aß(1-42) were isolated and purified. It was shown that the assembly of GroEL occurs without the addition of magnesium and potassium ions, as is commonly believed. According to the electron microscopy results, GroEL insignificantly affects the fibrillogenesis of the Aß(1-42) peptide, while α-crystallin prevents the elongation of the Aß(1-42) peptide fibrils. We have demonstrated that GroEL interacts nonspecifically with Aß(1-42), while α-crystallin does not interact with Aß(1-42) at all using surface plasmon resonance. CONCLUSION: The data obtained will help us understand the process of amyloid formation and the effect of various components on it.

Differentiation of Central Slovenian and Moscow populations of Rana temporaria frogs using peptide biomarkers of temporins family.

Skin secretion represents the only means of defense for the majority of frog species. That phenomenon is based on the fact that the main components of the secretion are peptides demonstrating greatly varying types of bioactivity. They fulfill regulatory functions, fight microorganisms and may be even helpful against predators. These peptides are considered to be rather promising pharmaceuticals of future generation as according to the present knowledge microorganisms are unlikely to develop resistance to them. Mass spectrometry sequencing of these peptides is the most efficient first step of their study providing reliably their primary structures, i.e., amino acids sequence and S-S bond motif. Besides discovering new bioactive peptides, mass spectrometry appears to be an efficient tool of taxonomy studies, allowing for distinguishing not only between closely related species, but also between populations of the same species. Application of several tandem mass spectrometry tools (CID, HCD, ETD, EThcD) available with Orbitrap mass analyzer allowed us to obtain full sequence of about 60 peptides in the secretion of Slovenian population of brown ranid frog Rana temporaria. The problem of sequence inside C-terminal cycle formed by two Cys and differentiation of isomeric Leu and Ile residues was done in top-down mode without any derivatization steps. Besides general biomarkers of Rana temporaria species, Central Slovenian population of Rana temporaria demonstrates six novel temporins and one brevinin 1, which may be treated as biomarkers of that population.

[Antibacterial effects of peptides synthesized based on the sequence of ribosome protein S1]. / Antibakterial'nye éffekty peptidov, sintezirovannykh na osnove posledovatel'nosti ribosomnogo belka S1.

Antibiotic resistance of bacteria is a topical problem on a global scale. Sometimes vigorous human activity leads to an increase in the number of bacteria carrying resistance genes in the environment. Antimicrobial peptides (AMPs) and similar compounds are potential candidates for combating antibiotic-resistant bacteria. Previously, we proposed and successfully tested on Thermus thermophilus a new mechanism of AMP action. This mechanism of directed coaggregation is based on the interaction of a peptide capable of forming fibrils with a target protein. In this work, we discuss the criteria for choosing a target for the targeted action of AMP, describe the features of the "parental" S1 ribosomal proteins T. thermophilus and Escherichia coli and the studied peptides using bioinformatic analysis methods, assess the antimicrobial effect of the synthesized peptides on a model organism of E. coli and cytotoxicity on cells of human fibroblasts. The obtained results will be important for the creation of new AMPs for pathogenic organisms.

Comparative Analysis of Aggregation of Thermus thermophilus Ribosomal Protein bS1 and Its Stable Fragment.

Functionally important multidomain bacterial protein bS1 is the largest ribosomal protein of subunit 30S. It interacts with both mRNA and proteins and is prone to aggregation, although this process has not been studied in detail. Here, we obtained bacterial strains overproducing ribosomal bS1 protein from Thermus thermophilus and its stable fragment bS1(49) and purified these proteins. Using fluorescence spectroscopy, dynamic light scattering, and high-performance liquid chromatography combined with mass spectrometric analysis of products of protein limited proteolysis, we demonstrated that disordered regions at the N- and C-termini of bS1 can play a key role in the aggregation of this protein. The truncated fragment bS1(49) was less prone to aggregation compared to the full-size bS1. The revealed properties of the studied proteins can be used to obtain protein crystals for elucidating the structure of the bS1 stable fragment.

Identification of Amyloidogenic Regions in the Spine of Insulin Fibrils.

To reveal conformational changes resulting in the formation of insulin fibrils, it is necessary to identify amyloidogenic regions in the structure of protein monomers. Different models of insulin fibrillogenesis have been proposed previously. However, precise regions responsible for the formation of amyloid fibrils have not been identified. Using bioinformatics programs for predicting amyloidogenic regions, we have determined some common amyloidogenic sequences in the structure of insulin monomers. The use of limited proteolysis and mass spectrometry analysis of the obtained protein fragments resistant to the action of proteases allowed us to identify amino acid sequences in the insulin structure that can form the spine of the insulin fibrils. The obtained results are in agreement with the earlier proposed model of fibril formation from the ring-like oligomers and can be used for designing insulin analogs resistant to amyloidogenesis.

[Amyloid Core Wild-Type Apomyoglobin and Its Mutant Variants Is Formed by Different Regions of the Polypeptide Chain].

As has been recently shown, the toxicity of protein aggregates is determined by their structure. Therefore, special attention has been focused on the search for factors that specify the structural features of formed amyloid fibrils. The effect of amino acid substitutions in apomyoglobin on the structural characteristics of its amyloid aggregates has been analyzed. The morphology and secondary structure of amyloids of the wild-type protein and its mutant variants Val10Ala, Val10Phe, and Trp14Phe have been compared, and the regions involved in intermolecular interactions in fibrils have been determined using limited proteolysis and mass spectrometry. No considerable differences have been found in the morphology (shape, length, or diameter) or the content (percentage) of the cross-ß structure of apomyoglobin amyloids and its mutant variants. Amyloid cores of wild-type apomyoglobin and variants with Val10Phe and Trp14Phe substitutions have been formed by different regions of the polypeptide chain. The case study of apomyoglobin demonstrates that the location of amyloidogenic regions in the polypeptide chain of wild-type protein and its mutant forms can differ. Thus, possible structural changes in amyloids resulting from amino acid substitutions should be taken into account when studying phenotype aggregation.

[Search for Functionally Significant Motifs and Amino Acid Residues of Actin].

The scientific interest to the structural and functional properties of actin is determined by its abundance in cells. Being an important component of the cytoskeleton, actin is involved in many protein-protein interactions. Using crystal structures and molecular models, we have mapped the amino acid residues that are involved in these interactions and form the ATP-binding site of the actin monomer. Moreover, using mass spectrometry and high-performance liquid chromatography methods, we have discovered the regions of the amino acid sequence of actin that form the core of the actin fibril. According to the bioinformatic analysis, these regions are amyloidogenic and are located in the C-terminal region and in the hinge between the first and third subdomains. The data obtained are applicable to chordate actin, because multiple alignment revealed highly conserved amino acid sequences. In turn, the comparison of the chordate actin with the bacterial homologs showed the presence of numerous amino acid substitutions and insertions.

[Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States].

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4-6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.

Characterization of biosurfactants produced by the oil-degrading bacterium Rhodococcus erythropolis S67 at low temperature.

Production of trehalolipid biosurfactants by Rhodococcus erythropolis S67 depending on the growth temperature was studied. R. erythropolis S67 produced glycolipid biosurfactants such as 2,3,4-succinoyl-octanoyl-decanoyl-2'-decanoyl trehalose and 2,3,4-succinoyl-dioctanoyl-2'-decanoyl trehalose during the growth in n-hexadecane medium at 26 and 10 °C, despite the different aggregate state of the hydrophobic substrate at low temperature. The surface tension of culture medium was found being reduced from 72 to 27 and 45 mN m-1, respectively. Production of trehalolipid biosurfactants by R. erythropolis S67 at low temperature could be useful for the biodegradation of petroleum hydrocarbons at low temperatures by enhancing the bioremediation performance in cold regions.

Peptide Aß(16-25) Forms Nanofilms in the Process of Its Aggregation.

A method for the synthesis and high purification of fragments of Aß(1-42) peptide has been elaborated. We have synthesized the amyloidogenic fragment Aß(16-25) predicted by us and studied the process of its aggregation by electron microscopy and X-ray analysis. Electron microscopy images show that the peptide forms a film, which is not characteristic of amyloid fibrils. At the same time, according to the X-ray diffraction data, its preparations display the presence of two main reflections (4.6-4.8 and 8-12 Å) characteristic of cross-ß structure of amyloid fibrils. Thus, the fragment Aß(16-25) that we predicted is a promising object not only for studying the process of polymerization of the peptides/proteins, but also for using it as a nanomaterial to study a number of biological processes.

Determination of Regions Involved in Amyloid Fibril Formation for Aß(1-40) Peptide.

The studies of amyloid structures and the process of their formation are important problems of biophysics. One of the aspects of such studies is to determine the amyloidogenic regions of a protein chain that form the core of an amyloid fibril. We have theoretically predicted the amyloidogenic regions of the Aß(1-40) peptide capable of forming an amyloid structure. These regions are from 16 to 21 and from 32 to 36 amino acid residues. In this work, we have attempted to identify these sites experimentally by the method of tandem mass spectrometry. As a result, we show that regions of the Aß(1-40) peptide from 16 to 22 and from 28 to 40 amino acid residues are resistant to proteases, i.e. they are included in the core of amyloid fibrils. Our results correlate with the results of the theoretical prediction.

Determination of Size of Folding Nuclei of Fibrils Formed from Recombinant Aß(1-40) Peptide.

We have developed a highly efficient method for purification of the recombinant product Aß(1-40) peptide. The concentration dependence of amyloid formation by recombinant Aß(1-40) peptide was studied using fluorescence spectroscopy and electron microscopy. We found that the process of amyloid formation is preceded by lag time, which indicates that the process is nucleation-dependent. Further exponential growth of amyloid fibrils is followed by branching scenarios. Based on the experimental data on the concentration dependence, the sizes of the folding nuclei of fibrils were calculated. It turned out that the size of the primary nucleus is one "monomer" and the size of the secondary nucleus is zero. This means that the nucleus for new aggregates can be a surface of the fibrils themselves. Using electron microscopy, we have demonstrated that fibrils of these peptides are formed by the association of rounded ring structures.

Comparison of experimental and theoretical data on hydrogen-deuterium exchange for ten globular proteins.

The number of protons available for hydrogen-deuterium exchange was predicted for ten globular proteins using a method described elsewhere by the authors. The average number of protons replaced by deuterium was also determined by mass spectrometry of the intact proteins in their native conformations. Based on these data, we find that two models proposed earlier agree with each other in estimation of the number of protons replaced by deuterium. Using a model with a probability scale for hydrogen bond formation, we estimated a number of protons replaced by deuterium that is close to the experimental data for long-term incubation in D(2)O (24 h). Using a model based on estimations with a scale of the expected number of contacts in globular proteins there is better agreement with the experimental data obtained for a short period of incubation in D(2)O (15 min). Therefore, the former model determines weakly fluctuating parts of a protein that are in contact with solvent only for a small fraction of the time. The latter model (based on the scale of expected number of contacts) predicts either flexible parts of a protein chain exposed to interactions with solvent or disordered parts of the protein.

Optimal region of average side-chain entropy for fast protein folding.

Search and study the general principles that govern kinetics and thermodynamics of protein folding generates new insight into the factors that control this process. Here, we demonstrate based on the known experimental data and using theoretical modeling of protein folding that side-chain entropy is one of the general determinants of protein folding. We show for proteins belonging to the same structural family that there exists an optimal relationship between the average side-chain entropy and the average number of contacts per residue for fast folding kinetics. Analysis of side-chain entropy for proteins that fold without additional agents demonstrates that there exists an optimal region of average side-chain entropy for fast folding. Deviation of the average side-chain entropy from the optimal region results in an anomalous protein folding process (prions, alpha-lytic protease, subtilisin, some DNA-binding proteins). Proteins with high or low side-chain entropy would have extended unfolded regions and would require some additional agents for complete folding. Such proteins are common in nature, and their structure properties have biological importance.

[Monomeric form of the molecular chaperone GroEL: structure, stability, and oligomerization]. / Monomernaia forma molekuliarnogo shaperona GroEL: struktura, stabil'nost' i oligomerizatsiia.

The structure and stability in solution of the monomeric form of GroEL were studied by the methods of circular dichroism, binding of a hydrophobic probe, limited proteolysis, modification of thiol groups, sedimentation, and size-exclusion chromatography. The monomeric GroEL at 23 degrees C was shown to be a globular protein with a pronounced secondary and a rigid tertiary structure. It exhibited no marked tendency to oligomerization in the absence of adenine nucleotides. However, the free monomeric GroEL was substantially less stable to urea and heat than the corresponding subunit in the composition of native oligomeric particles. The monomeric form also bound the hydrophobic probe, 8-anilino-1-naphthalenesulfonic acid, by an order of magnitude better than the subunit in the oligomeric particles. The ATP-induced oligomerization process of both folded and unfolded GroEL monomers was studied. The oligomerization rate was found to be the same for both monomers, and, therefore, should be limited by the ATP-dependent "arrangement" of the sites in the folded monomers responsible for the oligomerization rather than by the spontaneous refolding of monomers.

Green fluorescent protein purification by organic extraction.

Green fluorescent protein (GFP) is widely used as an excellent reporter molecule in biochemistry and cell biology. Some biochemical and immunological assays require high-purity GFP. However, the majority of current procedures for GFP purification include multiple time-consuming chromatography steps with a low yield of the desired product or require tag-containing proteins. An alternative method is described for the GFP purification without affinity extensions using organic extraction yielding a highly homogeneous protein indistinguishable in spectroscopic properties from that purified by previous methods.

[Denatured transitions of the molecular chaperone GroEL from Escherichia coli]. / Denaturatsionnye perekhody molekulianogo shaperona GroEL iz Escherichiacoli.

Conformational changes of oligomeric particle of GroEL chaperone from E. coli in solution were studied, which proceed during its denaturation upon the action of elevated urea concentration, temperature, and extremal pH values by the methods of CD, light scattering, scanning microcalorimetry, hydrophobic probe binding, and ATPase activity measurements. The ranges of changing the external conditions; within which GroEL retains its structure and functions, were determined. Denaturation transitions were found to be cooperative, pronounced, and irreversible. In the pH range from 6.0 to 9.6, the three-step change of the ATPase activity of GroEL was shown to occur with half-transition pH1/2 of 6.3, 8.5, and 9.3. It does not result in any essential structural changes and is probably associated with a protonation/deprotonation of amino acid residues important for the GroEL ATPase activity.

Ligands regulate GroEL thermostability.

Escherichia coli heat-shock proteins GroEL and GroES stimulate (in an ATP-dependent manner) the folding of various proteins. In this study scanning microcalorimetry was applied to investigate GroEL thermostability in the presence of its ligands. Mg2+ and K+ ions stabilize while ADP destabilizes the GroEL molecule against the action of temperature. Furthermore, ADP essentially increases the number of binding sites for the hydrophobic probe (ANS) and the number of GroEL SH-groups accessible to Ellman's reagent as well as the accessibility of the protein to the action of trypsin. The interaction of GroEL with GroES in the presence of Mg2+-ADP eliminates the destabilizing effect of ADP on the GroEL molecule against the action of temperature and Ellman's reagent but does not change its hydrophobicity and accessibility to trypsin.

[Denatured state of lysozyme in dimethyl sulfoxide]. / Denaturirovannoe sostoianie lizotsima v dimetilsul'fokside.

An influence of DMSO on lysozyme structure in solution was studied by fluorescence and optical rotary dispersion methods. Change in the protein structure was shown to proceed at DMSO concentration in water greater than 60% and result in an increase of the protein helicity. However, this structural state of lysozyme is similar to that of the unstructured peptides obtained by its complete proteolysis and is characterized by parameters of accessibility to solvent and mobility of their intrinsic chromophores. The data obtained evidenced that long range interactions have a little influence on the maintenance of the residual secondary structure of lysozyme in the presence of DMSO.