How proteins manage to fold and how chaperones manage to assist the folding.

This review presents the current understanding of (i) spontaneous self-organization of spatial structures of protein molecules, and (ii) possible ways of chaperones' assistance to this process. Specifically, we overview the most important features of spontaneous folding of proteins (mostly, of the single-domain water-soluble globular proteins): the choice of the unique protein structure among zillions of alternatives, the nucleation of the folding process, and phase transitions within protein molecules. We consider the main experimental facts on protein folding, both in vivo and in vitro, of both kinetic and thermodynamic nature.

Acceleration of carbonic anhydrase amyloid aggregation leads to a decrease in the fibrils toxicity.

Cells damage by protein aggregates is one of the causes of amyloid diseases. This study aimed to explore the structural features of cytotoxic amyloid fibrils and to find strategies to reduce their damaging effect. Bovine carbonic anhydrase B (BCAB) was chosen for this work due to high toxicity of its amyloid fibrils.

Co-chaperonin GroES subunit exchange as dependent on time, pH, protein concentration, and urea.

The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel.

Bacteriophage T5 dUTPase: Combination of Common Enzymatic and Novel Functions.

The main function of dUTPases is to regulate the cellular levels of dUTP and dTTP, thereby playing a crucial role in DNA repair mechanisms. Despite the fact that mutant organisms with obliterated dUTPase enzymatic activity remain viable, it is not possible to completely knock out the gene due to the lethal consequences of such a mutation for the organism. As a result, it is considered that this class of enzymes performs an additional function that is essential for the organism's survival.

Influence of Amino Acid Substitutions in ApoMb on Different Stages of Unfolding of Amyloids.

To date, most research on amyloid aggregation has focused on describing the structure of amyloids and the kinetics of their formation, while the conformational stability of fibrils remains insufficiently explored. The aim of this work was to investigate the effect of amino acid substitutions on the stability of apomyoglobin (ApoMb) amyloids. A study of the amyloid unfolding of ApoMb and its six mutant variants by urea has been carried out.

The New Functional Hybrid Chaperone Protein ADGroEL-SacSm.

The creation of new proteins by combining natural domains is a commonly used technique in protein engineering. In this work, we have tested the possibilities and limitations of using circular homo-oligomeric Sm-like proteins as a basis for attaching other domains. Attachment to such a stable base should bring target domains together and keep them in the correct mutual orientation.

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties.

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein enhanced green fluorescent protein (EGFP) or luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL-EGFP is evenly distributed within normally divided cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II.

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well.

Recombinant Globular Domain of TcpA Pilin from El Tor: Recovery from Inclusion Bodies and Structural Characterization.

The production of recombinant proteins in cells is often hampered by aggregation of newly synthesized proteins and formation of inclusion bodies. Here we propose the use of transverse urea gradient electrophoresis (TUGE) in testing the capability of folding of a recombinant protein from inclusion bodies dissolved in urea. A plasmid encoding the amino acid sequence 55-224 of TcpA pilin (C-terminal globular domain: TcpA-C) from El Tor enlarged by a His-tag on its N-terminus was expressed in cells.

The Denaturant- and Mutation-Induced Disassembly of Hexameric Hfq Y55W Mutant.

Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qβ host factor (Hfq) from mesophilic (). Using intrinsic tryptophan fluorescence, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC), we show that the dissociation of Hfq Y55W occurs either under the effect of GuHCl or during the pre-denaturing transition, when the protein concentration is decreased, with both events proceeding through the accumulation of stable intermediate states.

Carbonic anhydrase amyloid fibrils composed of laterally associated protofilaments show reduced cytotoxicity.

Cytotoxicity of amyloid fibrils has been shown to depend on their structure. However, specific features of toxic and non-toxic amyloids remain unclear. Here we focus on the relationship between structural characteristics of the fibrils and their cytotoxicity.

Under Conditions of Amyloid Formation Bovine Carbonic Anhydrase B Undergoes Fragmentation by Acid Hydrolysis.

The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins' aggregation remain insufficiently investigated. Our work aimed to research the amyloid aggregation of a large model protein, bovine carbonic anhydrase B (BCAB).

β-elimination of hyaluronate by red king crab hyaluronidase.

Crustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we show that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed that the molecular weight of a protein with hyalorunidase activity is 40-50 kDa.

The presence of cross-β-structure as a key determinant of carbonic anhydrase amyloid fibrils cytotoxicity.

In most cases high cytotoxicity is characteristic of aggregates formed during lag phase of amyloid formation, whereas mature fibrils represent the depot of protein molecules incapable of damaging cell membranes. However, new experimental data show that in cases of some proteins the fibrils are the most toxic type of aggregates. Meanwhile, structural characteristics of cytotoxic fibrils and mechanisms of their cell damaging action are insufficiently explored.

Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution.

The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL.

Phylogenetically Defined Isoforms of Invasion Factor InlB Differently Activate Intracellular Signaling Pathways and Interact with the Receptor gC1q-R.

The pathogenic Gram-positive bacterium has been evolving into a few phylogenetic lineages. Phylogenetically defined substitutions were described in the virulence factor InlB, which mediates active invasion into mammalian cells via interactions with surface receptors c-Met and gC1q-R. InlB internalin domain (idInlB) is central to interactions with c-Met.

Molecular Size Distribution of Fluorophores in Aquatic Natural Organic Matter: Application of HPSEC with Multi-Wavelength Absorption and Fluorescence Detection Following LPSEC-PAGE Fractionation.

Analytical high performance size exclusion liquid chromatography (HPSEC) with multiwavelength absorbance and fluorescence detections was used for the analysis of molecular size distribution and optical properties of dissolved natural organic matter. Experiments were conducted on Suwannee River organic matter (SRNOM) and its fractions A, B, C+D preliminary obtained by combination of preparative low pressure size exclusion chromatography and polyacrylamide gel electrophoresis (LPSEC-PAGE) and purified by dialysis on membrane with nominal cutoff 10 kDa, the fractions molecular size varied in order A > B > C + D > 10 kDa. The multistep fractionation of SRNOM enabled the size-separation of at least five types of humic-like fluorophores within NOM showing emission maxima at 465, 450, 435, 420, and 405 nm.

sw ApoMb Amyloid Aggregation under Nondenaturing Conditions: The Role of Native Structure Stability.

Investigation of the molecular mechanisms underlying amyloid-related human diseases attracts close attention. These diseases, the number of which currently is above 40, are characterized by formation of peptide or protein aggregates containing a cross-β structure. Most of the amyloidogenesis mechanisms described so far are based on experimental studies of aggregation of short peptides, intrinsically disordered proteins, or proteins under denaturing conditions, and studies of amyloid aggregate formations by structured globular proteins under conditions close to physiological ones are still in the initial stage.

The Mechanism Underlying Amyloid Polymorphism is Opened for Alzheimer's Disease Amyloid-β Peptide.

It has been demonstrated using Aβ40 and Aβ42 recombinant and synthetic peptides that their fibrils are formed of complete oligomer ring structures. Such ring structures have a diameter of about 8-9 nm, an oligomer height of about 2- 4 nm, and an internal diameter of the ring of about 3-4 nm. Oligomers associate in a fibril in such a way that they interact with each other, overlapping slightly.

X-ray diffraction and electron microscopy data for amyloid formation of Aβ40 and Aβ42.

The data presented in this article are related to the research article entitled "One of the possible mechanisms of amyloid fibrils formation based on the sizes of primary and secondary folding nuclei of Aβ40 and Aβ42" (Dovidchenko et al., 2016) [1]. Aβ peptide is one of the most intensively studied amyloidogenic peptides.

Isoforms of green fluorescent protein differ from each other in solvent molecules 'trapped' inside this protein.

Green fluorescent protein (GFP) has been studied quite thoroughly, however, up to now some experimental data have not been explained explicitly. For example, under native conditions this protein can have two isoforms differing in their mobility in gel. In this case, no differences between the isoforms are revealed under denaturing conditions.