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.

Verification of the Stabilized Protein Design Based on the Prediction of Intrinsically Disordered Regions: Ribosomal Proteins L1.

In our previous papers, we proposed the idea that programs predicting intrinsically disordered regions in amino acid sequences can be used for finding weakened sites in proteins. The regions predicted by such programs are suitable targets for the introduction of protein-stabilizing mutations. However, for each specific protein, it remains unclear what determines protein stabilization - the amino acid sequence (and accordingly, prediction of weakened sites) or the 3D structure. To answer this question, it is necessary to study two proteins with similar structures but different amino acid sequences and, consequently, different predictions of weakened regions. By introducing identical mutations into identical elements of the two proteins, we will be able to reveal whether predictions of the weakened sites or the 3D protein structure are the key factors in the protein stability increase. Here, we have chosen ribosomal proteins L1 from the halophilic archaeon Haloarcula marismortui (HmaL1) and extremophilic bacterium Aquifex aeolicus (AaeL1). These proteins are identical in their structure but different in amino acid sequences. A disulfide bond introduced into the region predicted as the structured one in AaeL1 did not lead to the increase in the protein melting temperature. At the same time, a disulfide bond introduced into the same region in HmaL1 that was predicted as a weakened one, resulted in the increase in the protein melting temperature by approximately 10°C.

Disruption of flavin homeostasis in isolated rat liver mitochondria.

It has been shown that spontaneous release of non-covalent flavins (from flavoenzymes) begins after isolation of mitochondria from rat liver, which is hydrolyzed to riboflavin. This process is stopped by 1 mM EDTA in the incubation medium. In the presence of NADH, deflavinization of flavoproteins leads to formation of superoxide by at least of three processes. The first of these occurs in complex I as a result of the spontaneous release of FMN from the active center. This process is inhibited by adenosine and guanosine phosphates, as well as NAD, but amplified by nicotinamide. The second process is associated with enzymatic hydrolysis of FAD and FMN to riboflavin; it is blocked by EDTA, AMP, NA, NAD. The third process is associated with non-enzymatic hydrolysis of FAD by iron ions in matrix; it is blocked by EDTA and AMP.

The binding of monomeric amyloid ß peptide to serum albumin is affected by major plasma unsaturated fatty acids.

Human serum albumin (HSA) serves as a natural depot of amyloid ß peptide (Aß). Improvement of Aß binding to HSA should impede Alzheimer's disease (AD). We developed a method for quantitation of the interaction between monomeric Aß40/42 and HSA using surface plasmon resonance spectroscopy. The dissociation constant of HSA complex with recombinant Aß40/42 is 0.2-0.3 µM. Flemish variant of Aß40 has 2.5-10-fold higher affinity to HSA. The parameters of the HSA-Aß interaction are selectively sensitive to HSA binding of major plasma unsaturated fatty acids and Cu2+. Linoleic and arachidonic acids promote the HSA-Aß42 interaction. The developed methodology for quantitation of HSA-Aß interaction may serve as a tool for search of compounds favoring HSA-Aß interaction, thereby preventing AD progression.

[Effect of Substitutions in Surface Amino Acid on Energy Profile of Apomyoglobin].

Studies on the process of spontaneous protein folding into a unique native state are an important issue of molecular biology. Apomyoglobin from the sperm whale is a convenient model for these studies in vitro. Here, we present the results of equilibrium and kinetic experiments carried out in a study on the folding and unfolding of eight mutant apomyoglobin forms of with hydrophobic amino acid substitutions on the protein surface. Calculated values of apparent constants of folding/unfolding rates, as well as the data on equilibrium conformational transitions in the urea concentration range of 0-6 М at 11°C are given. Based on the obtained information on the kinetic properties of the studied proteins, a Φ-value analysis of the transition state has been performed and values of urea concentrations corresponding to the midpoint of the transition from the native to intermediate state have been determined for the given forms of mutant apomyoglobin. It has been found that a significant increase in the stability of the native state can be achieved by a small number of amino acid substitutions on the protein surface. It has been shown that the substitution of only one amino acid residue exclusively affects the height of the energy barrier that separates different states of apomyoglobin.

[Limited Trypsinolysis of GroES: The Effect on the Interaction with GroEL and Assembly In Vitro].

GroES is a heptameric partner of tetradecameric molecular chaperone GroEL, which ensures the correct folding and assembly of numerous cellular proteins both in vitro and in vivo. This work demonstrates the results of a study of structural aspects of GroES that affect its interaction with GroEL and reassembly. The effect of limited trypsinolysis of GroES on these processes has been studied. It has been shown that limited trypsinolysis of GroES is only strongly pronounced outside the complex with GroEL and results in the cleavage of the peptide bond between Lys20 and Ser21. The N-terminal fragment (~2 kDa) is retained in the GroES particle, which maintains its heptaoligomeric structure but loses the ability to interact with GroEL and dissociates upon a change in the pH from 7 to 8. Trypsin-nicked GroES cannot reassemble after urea-induced unfolding, while the urea-induced unfolding of intact GroES is fully reversible. The reported results indicate the important role of the N-terminal part of GroES subunit in the assembly of its heptameric structure and the interaction with GroEL.

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.

Dataset concerning GroEL chaperonin interaction with proteins.

GroEL chaperonin is well-known to interact with a wide variety of polypeptide chains. Here we show the data related to our previous work (http://dx.doi.org/10.1016/j.pep.2015.11.020[1]), and concerning the interaction of GroEL with native (lysozyme, α-lactalbumin) and denatured (lysozyme, α-lactalbumin and pepsin) proteins in solution. The use of affinity chromatography on the base of denatured pepsin for GroEL purification from fluorescent impurities is represented as well.

Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

Molecular chaperone GroEL/ES: unfolding and refolding processes.

Molecular chaperones are a special class of heat shock proteins (Hsp) that assist the folding and formation of the quaternary structure of other proteins both in vivo and in vitro. However, some chaperones are complex oligomeric proteins, and one of the intriguing questions is how the chaperones fold. The representatives of the Escherichia coli chaperone system GroEL (Hsp60) and GroES (Hsp10) have been studied most intensively. GroEL consists of 14 identical subunits combined into two interacting ring-like structures of seven subunits each, while the co-chaperone GroES interacting with GroEL consists of seven identical subunits combined into a dome-like oligomeric structure. In spite of their complex quaternary structure, GroEL and GroES fold well both in vivo and in vitro. However, the specific oligomerization of GroEL subunits is dependent on ligands and external conditions. This review analyzes the literature and our own data on the study of unfolding (denaturation) and refolding (renaturation) processes of these molecular chaperones and the effect of ligands and solvent composition. Such analysis seems to be useful for understanding the folding mechanism not only of the GroEL/GroES complex, but also of other oligomeric protein complexes.

Multy-state protein: Determination of carbonic anhydrase free-energy landscape.

Studies of the folding pathway of large proteins whose kinetics is complicated due to the formation of several intermediate states are most frequently impeded or totally impossible because of rapid folding phase occurring during instrument dead time. In this paper the obtaining of energy characteristics of one of such proteins-carbonic anhydrase B-is reported. Tryptophan fluorescence and absorption methods have been used to measure the folding and unfolding kinetics of carbonic anhydrase B at different urea concentrations. In spite of the fact that the formation of the initial intermediate state of this protein takes place during the instrument dead time, the population of this state has been estimated in a wide range of urea concentrations. The use of the population of the rapidly formed intermediate state and the effective rates of slow phases of the protein folding/unfolding permitted us to calculate free energies of all the protein states and the height of energy barriers between them. It has been shown that folding of carbonic anhydrase B can be described by a consecutive reaction scheme. The possibility to obtain energy characteristics of carbonic anhydrase would allow studying structural characteristics of both intermediate and transition states via site-directed mutations.

Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

[The interaction of the GroEL chaperone with early kinetic intermediates of renaturing proteins inhibits the formation of their native structure]. / Vzaimodeistvie shaperona GroEL s rannimi kineticheskimi promezhutochnymi sostoianiiami renaturiruiushchikh belkov ingibiruet formirovanie ikh nativnoi struktury.

The main function of the chaperone GroEL is to prevent nonspecific association of nonnative protein chains and provide their correct folding. In the present work, the renaturation kinetics of three globular proteins (human alpha-lactalbumin, bovine carbonic anhydrase, and yeast phosphoglycerate kinase) in the presence of different molar excess of GroEL (up to 10-fold) was studied. It was shown that the formation of the native structure during the refolding of these proteins is retarded with an increase in GroEL molar excess due to the interaction of kinetic protein intermediates with the chaperone. Mg(2+)-ATP and Mg(2+)-ADP weaken this interaction and decrease the retarding effect of GroEL on the protein refolding kinetics. The theoretical modeling of protein folding in the presence of GroEL showed that the experimentally observed linear increase in the protein refolding half-time with increasing molar excess of GroEL must occur only when the protein adopts its native structure outside of GroEL (i.e. in the free state), while the refolding of the protein in the complex with GroEL is inhibited. The dissociation constants of GroEL complexed with the kinetic intermediates of the proteins studied were evaluated, and a simple mechanism of the functioning of GroEL as a molecular chaperone was proposed.

[Effect of ADP and GroES on interaction of molecular chaperonin GroEL with non-native lysozyme]. / Vliianie ADP i GroES na vzaimodeistvie molekuliarnogo shaperonina GroEL s nenativnym lizotsimom.

The interaction of the molecular chaperonin GroEL with fluorescein-labeled lysozyme in the presence of high concentrations of thiol reagent--dithiothreitol (DTT) has been studied. In case of high concentrations of DTT lysozyme loses the native conformation due to the disruption of the intramolecular disulfide bonds stabilizing its structure and effectively aggregates. It has been shown that in the presence of high concentrations of DTT and two-fold molar excess of GroEL the lysozyme tightly interacts with GroEL that essentially decreases the efficiency of its aggregation. The addition of ADP to the complex of GroEL with nonnative lysozyme noticeably decreases the interaction of the chaperonin with nonnative protein target resulting in some increase of the efficiency of its aggregation. However, the addition of the co-chaperonin GroES together with ADP (i.e. the formation of the complex of GroEL with GroES) leads to drastic weakness of the interaction of GroEL with nonnative lysozyme and the efficiency of its aggregation becomes comparable with that in the absence of GroEL.

[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.

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.