Broken but not beaten: Challenge of reducing the amyloids pathogenicity by degradation.

BACKGROUND: The accumulation of ordered protein aggregates, amyloid fibrils, accompanies various neurodegenerative diseases (such as Parkinson's, Huntington's, Alzheimer's, etc.) and causes a wide range of systemic and local amyloidoses (such as insulin, hemodialysis amyloidosis, etc.). Such pathologies are usually diagnosed when the disease is already irreversible and a large amount of amyloid plaques have accumulated. In recent years, new drugs aimed at reducing amyloid levels have been actively developed. However, although clinical trials have demonstrated a reduction in amyloid plaque size with these drugs, their effect on disease progression has been controversial and associated with significant side effects, the reasons of which are not fully understood. AIM OF REVIEW: The purpose of this review is to summarize extensive array of data on the effect of exogenous and endogenous factors (physico-mechanical effects, chemical effects of low molecular weight compounds, macromolecules and their complexes) on the structure and pathogenicity of mature amyloids for proposing future directions of the development of effective and safe anti-amyloid therapeutics. KEY SCIENTIFIC CONCEPTS OF REVIEW: Our analysis show that destruction of amyloids is in most cases incomplete and degradation products often retain the properties of amyloids (including high and sometimes higher than fibrils, cytotoxicity), accelerate amyloidogenesis and promote the propagation of amyloids between cells. Probably, the appearance of protein aggregates, polymorphic in structure and properties (such as amorphous aggregates, fibril fragments, amyloid oligomers, etc.), formed because of uncontrolled degradation of amyloids, may be one of the reasons for the ambiguous effectiveness and serious side effects of the anti-amyloid drugs. This means that all medications that are supposed to be used both for degradation and slow down the fibrillogenesis must first be tested on mature fibrils: the mechanism of drug action and cytotoxic, seeding, and infectious activity of the degradation products must be analyzed.

Structural determinants of odorant-binding proteins affecting their ability to form amyloid fibrils.

The formation of amyloid fibrils is associated with many severe pathologies as well as the execution of essential physiological functions by proteins. Despite the diversity, all amyloids share a similar morphology and consist of stacked ß-strands, suggesting high amyloidogenicity of native proteins enriched with ß-structure. Such proteins include those with a ß-barrel-like structure with ß-strands arranged into a cylindrical ß-sheet. However, the mechanisms responsible for destabilization of the native state and triggering fibrillogenesis have not thoroughly explored yet. Here we analyze the structural determinants of fibrillogenesis in proteins with ß-barrel structures on the example of odorant-binding protein (OBP), whose amyloidogenicity was recently demonstrated in vitro. We reveal a crucial role in the fibrillogenesis of OBPs for the "open" conformation of the molecule. This conformation is achieved by disrupting the interaction between the ß-barrel and the C-terminus of protein monomers or dimers, which exposes "sticky" amyloidogenic sites for interaction. The data suggest that the "open" conformation of OBPs can be induced by destabilizing the native ß-barrel structure through the disruption of: 1) intramolecular disulfide cross-linking and non-covalent contacts between the C-terminal fragment and ß-barrel in the protein's monomeric form, or 2) intermolecular contacts involved in domain swapping in the protein's dimeric form.

Prediction of the Feasibility of Using the ≪Gold Standard≫ Thioflavin T to Detect Amyloid Fibril in Acidic Media.

Ordered protein aggregates, amyloid fibrils, form toxic plaques in the human body in amyloidosis and neurodegenerative diseases and provide adaptive benefits to pathogens and to reduce the nutritional value of legumes. To identify the amyloidogenic properties of proteins and study the processes of amyloid fibril formation and degradation, the cationic dye thioflavin T (ThT) is the most commonly used. However, its use in acidic environments that induce amyloid formation in vitro can sometimes lead to misinterpretation of experimental results due to electrostatic repulsion. In this work, we show that calculating the net charge per residue of amyloidogenic proteins or peptides is a simple and effective approach for predicting whether their fibrils will interact with ThT at acidic pH. In particular, it was shown that at pH 2, proteins and peptides with a net charge per residue > +0.18 are virtually unstained by this fluorescent probe. The applicability of the proposed approach was demonstrated by predicting and experimentally confirming the absence of ThT interaction with amyloids formed from green fluorescent (sfGFP) and odorant-binding (bOBP) proteins, whose fibrillogenesis was first carried out in an acidic environment. Correct experimental evidence that the inability to detect these fibrils under acidic conditions is precisely because of the lack of dye binding to amyloids (and not their specific structure or the low fluorescence quantum yield of the bound dye) and that the number of ThT molecules associated with fibrils increases with decreasing acidity of the medium was obtained by using the equilibrium microdialysis approach.

Mammalian odorant-binding proteins are prone to form amorphous aggregates and amyloid fibrils.

Odorant-binding proteins are involved in perceiving smell by capturing odorants within the protein's ß-barrel. On the example of bovine odorant-binding protein (bOBP), the structural organization of such proteins and their ability to bind ligands under various conditions in vitro were examined. We found a tendency of bOBP to form oligomers and small amorphous aggregates without disturbing the integrity of protein monomers at physiological conditions. Changes in environmental parameters (increased temperature and pH) favored the formation of larger and dense supramolecular complexes that significantly reduce the binding of ligands by bOBP. The ability of bOBP to form fibrillar aggregates with the properties of amyloids, including high cytotoxicity, was revealed at sample stirring (even at physiological temperature and pH), at medium acidification or pre-solubilization with hexafluoroisopropanol. Fibrillogenesis of bOBP was initiated by the dissociation of the protein's supramolecular complexes into monomers and the destabilization of the protein's ß-barrels without a significant destruction of its native ß-strands.

Amyloid fibrils degradation: the pathway to recovery or aggravation of the disease?

Background: The most obvious manifestation of amyloidoses is the accumulation of amyloid fibrils as plaques in tissues and organs, which always leads to a noticeable deterioration in the patients' condition and is the main marker of the disease. For this reason, early diagnosis of amyloidosis is difficult, and inhibition of fibrillogenesis, when mature amyloids are already accumulated in large quantities, is ineffective. A new direction for amyloidosis treatment is the development of approaches aimed at the degradation of mature amyloid fibrils. In the present work, we investigated possible consequences of amyloid's degradation. Methods: We analyzed the size and morphology of amyloid degradation products by transmission and confocal laser scanning microscopy, their secondary structure and spectral properties of aromatic amino acids, intrinsic chromophore sfGFP, and fibril-bound amyloid-specific probe thioflavin T (ThT) by the absorption, fluorescence and circular dichroism spectroscopy, as well as the cytotoxicity of the formed protein aggregates by MTT-test and their resistance to ionic detergents and boiling by SDS-PAGE. Results: On the example of sfGFP fibrils (model fibrils, structural rearrangements of which can be detected by a specific change in the spectral properties of their chromophore), and pathological Aß-peptide (Aß42) fibrils, leading to neuronal death in Alzheimer's disease, the possible mechanisms of amyloids degradation after exposure to factors of different nature (proteins with chaperone and protease activity, denaturant, and ultrasound) was demonstrated. Our study shows that, regardless of the method of fibril degradation, the resulting species retain some amyloid's properties, including cytotoxicity, which may even be higher than that of intact amyloids. Conclusion: The results of our work indicate that the degradation of amyloid fibrils in vivo should be treated with caution since such an approach can lead not to recovery, but to aggravation of the disease.

Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties.

Understanding the photophysical properties and stability of near-infrared fluorescent proteins (NIR FPs) based on bacterial phytochromes is of great importance for the design of efficient fluorescent probes for use in cells and in vivo. Previously, the natural ligand of NIR FPs biliverdin (BV) has been revealed to be capable of covalent binding to the inherent cysteine residue in the PAS domain (Cys15), and to the cysteine residue introduced into the GAF domain (Cys256), as well as simultaneously with these two residues. Here, based on the spectroscopic analysis of several NIR FPs with both cysteine residues in PAS and GAF domains, we show that the covalent binding of BV simultaneously with two domains is the reason for the higher quantum yield of BV fluorescence in these proteins as a result of rigid fixation of the chromophore in their chromophore-binding pocket. We demonstrate that since the attachment sites are located in different regions of the polypeptide chain forming a figure-of-eight knot, their binding to BV leads to shielding of many sites of proteolytic degradation due to additional stabilization of the entire protein structure. This makes NIR FPs with both cysteine residues in PAS and GAF domains less susceptible to cleavage by intracellular proteases.

sfGFP throws light on the early stages of ß-barrel amyloidogenesis.

The accumulation of ß-sheet-rich protein aggregates, amyloid fibrils, accompanies severe pathologies (Alzheimer's, Parkinson's diseases, ALS, etc.). The high amyloidogenicity of proteins with a native ß-barrel structure, and the amyloidogenic peptides ability to form a universal cylindrin-like oligomeric state were proven. The mechanisms for the proteins' transformation from this state to a fibrillar one are still not fully understood. We defined the structural rearrangements of the amyloidogenic ß-barrel superfolder GFP (sfGFP) prior to fibrillogenesis using its tryptophan and chromophore fluorescence. We characterized the early intermediate "native-like" state preserving the integrity of the sfGFP ß-barrel scaffold despite the partial distortion of the three ß-strands closing it. The interaction between the "melted" regions of the protein leads to the assembly of high molecular weight complexes, which are not dynamic structures but are less stable and less cytotoxic than mature amyloids. Additional contacts of sfGFP monomers facilitate the global reorganization of its structure and stabilization of the second intermediate state in which the ß-barrel opens and some of the native α-helices and disordered regions refold into non-native ß-strands, which, along with native ß-strands, form an amyloid fiber. Reported sfGFP structural transformations may occur during the fibrillogenesis of other ß-barrel proteins, and the identified intermediate states are likely universal. Thus sfGFP can be used as a sensing platform to develop therapeutic agents inhibiting amyloidogenesis through interaction with protein intermediates and destroying low-stable aggregates formed at the early stages of fibrillogenesis.

Liquid-liquid phase separation as an organizing principle of intracellular space: overview of the evolution of the cell compartmentalization concept.

At the turn of the twenty-first century, fundamental changes took place in the understanding of the structure and function of proteins and then in the appreciation of the intracellular space organization. A rather mechanistic model of the organization of living matter, where the function of proteins is determined by their rigid globular structure, and the intracellular processes occur in rigidly determined compartments, was replaced by an idea that highly dynamic and multifunctional "soft matter" lies at the heart of all living things. According this "new view", the most important role in the spatio-temporal organization of the intracellular space is played by liquid-liquid phase transitions of biopolymers. These self-organizing cellular compartments are open dynamic systems existing at the edge of chaos. They are characterized by the exceptional structural and compositional dynamics, and their multicomponent nature and polyfunctionality provide means for the finely tuned regulation of various intracellular processes. Changes in the external conditions can cause a disruption of the biogenesis of these cellular bodies leading to the irreversible aggregation of their constituent proteins, followed by the transition to a gel-like state and the emergence of amyloid fibrils. This work represents a historical overview of changes in our understanding of the intracellular space compartmentalization. It also reflects methodological breakthroughs that led to a change in paradigms in this area of science and discusses modern ideas about the organization of the intracellular space. It is emphasized here that the membrane-less organelles have to combine a certain resistance to the changes in their environment and, at the same time, show high sensitivity to the external signals, which ensures the normal functioning of the cell.

New Evidence of the Importance of Weak Interactions in the Formation of PML-Bodies.

In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4-6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML-/-), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid-liquid phase separation (LLPS) leading to the formation of PML bodies.

ß-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis.

Insoluble protein aggregates with fibrillar morphology called amyloids and ß-barrel proteins both share a ß-sheet-rich structure. Correctly folded ß-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another-followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that ß-barrel proteins can adopt cross-ß amyloid folds have emerged. Different ß-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by ß-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with ß-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the ß-barrel topology. This state can be intermediate on the pathway of fibrillogenesis ("on-pathway state"), or can be formed as a result of an alternative assembly of partially unfolded monomers ("off-pathway state"). The ß-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming ß-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only "the tip of the iceberg". Here, we summarize the data on the amyloid-forming ß-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of ß-folds.

Photophysical Properties of BADAN Revealed in the Study of GGBP Structural Transitions.

The fluorescent dye BADAN (6-bromoacetyl-2-dimetylaminonaphtalene) is widely used in various fields of life sciences, however, the photophysical properties of BADAN are not fully understood. The study of the spectral properties of BADAN attached to a number of mutant forms of GGBP, as well as changes in its spectral characteristics during structural changes in proteins, allowed to shed light on the photophysical properties of BADAN. It was shown that spectral properties of BADAN are determined by at least one non-fluorescent and two fluorescent isomers with overlapping absorbing bands. It was found that BADAN fluorescence is determined by the unsolvated "PICT" (planar intramolecular charge transfer state) and solvated "TICT" (twisted intramolecular charge transfer state) excited states. While "TICT" state can be formed both as a result of the "PICT" state solvation and as a result of light absorption by the solvated ground state of the dye. BADAN fluorescence linked to GGBP/H152C apoform is quenched by Trp 183, but this effect is inhibited by glucose intercalation. New details of the changes in the spectral characteristics of BADAN during the unfolding of the protein apo and holoforms have been obtained.

New findings on GFP-like protein application as fluorescent tags: Fibrillogenesis, oligomerization, and amorphous aggregation.

Green fluorescent proteins (GFP) are commonly used as fluorescent tags and biosensors in cell biology and medicine. However, the propensity of GFP-like proteins to aggregate and the consequence of intermolecular interaction for their application have not been thoroughly examined. In this work, alternative aggregation pathways of superfolder green fluorescent protein (sfGFP) were demonstrated using a spectroscopic and microscopic study of the samples prepared by equilibrium microdialysis. Besides oligomerization of native monomers, we showed for the first time the condition-specific formation by sfGFP of either amyloid fibrils (at increased temperature or acidity) or amorphous aggregates (at physiological conditions). Both types of sfGFP aggregates had lost green fluorescence and were toxic to cells. Thus, when using GFP-like proteins as fluorescent tags, one should take into account their high ability to form aggregates with lost unique visible fluorescence in the cellular environment, which affects cell viability. Moreover, the results of this work cast doubt on the correctness of the data on the fibrillogenesis of various amyloidogenic proteins obtained using their fusion with GFP-like proteins.

Trypsin Induced Degradation of Amyloid Fibrils.

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

Photo-dependent membrane-less organelles formed from plant phyB and PIF6 proteins in mammalian cells.

Plant photobodies are the membrane-less organelles (MLOs) that can be generated by protein-protein interactions between active form of phytochrome B (phyB) and phytochrome-interacting factors (PIFs). These organelles regulate plant photomorphogenesis. In this study, we developed two chimeric proteins with fluorescent proteins, phyB fused to EGFP and PIF6 fused to mCherry, and investigated their exogenous expression in mammalian cells by confocal fluorescence microscopy. Results showed that irradiation with diffused 630-nm light induced formation and subsequent increase in sizes of the MLOs. The assembly and disassembly of the photo-inducible MLOs in the mammalian cell cytoplasm obeyed the laws inherent in the concentration-dependent phase separation of biopolymers. The sizes of MLOs formed from phyB and PIF6 in mammalian cells corresponded to the sizes of the so-called "early" photobodies in plant cells. These results suggested that the first step for the formation of plant photobodies might be based on the light-dependent liquid-liquid phase separation of PIFs and other proteins that can specifically interact with the active form of phyB. The developed chimeric proteins in principle can be used to control the assembly and disassembly of photo-inducible MLOs, and thereby to regulate various intracellular processes in mammalian cells.

Alpha-B-Crystallin Effect on Mature Amyloid Fibrils: Different Degradation Mechanisms and Changes in Cytotoxicity.

Given the ability of molecular chaperones and chaperone-like proteins to inhibit the formation of pathological amyloid fibrils, the chaperone-based therapy of amyloidosis has recently been proposed. However, since these diseases are often diagnosed at the stages when a large amount of amyloids is already accumulated in the patient's body, in this work we pay attention to the undeservedly poorly studied problem of chaperone and chaperone-like proteins' effect on mature amyloid fibrils. We showed that a heat shock protein alpha-B-crystallin, which is capable of inhibiting fibrillogenesis and is found in large quantities as a part of amyloid plaques, can induce degradation of mature amyloids by two different mechanisms. Under physiological conditions, alpha-B-crystallin induces fluffing and unweaving of amyloid fibrils, which leads to a partial decrease in their structural ordering without lowering their stability and can increase their cytotoxicity. We found a higher correlation between the rate and effectiveness of amyloids degradation with the size of fibrils clusters rather than with amino acid sequence of amyloidogenic protein. Some external effects (such as an increase in medium acidity) can lead to a change in the mechanism of fibrils degradation induced by alpha-B-crystallin: amyloid fibers are fragmented without changing their secondary structure and properties. According to recent data, fibrils cutting can lead to the generation of seeds for new bona fide amyloid fibrils and accelerate the accumulation of amyloids, as well as enhance the ability of fibrils to disrupt membranes and to reduce cell viability. Our results emphasize the need to test the chaperone effect not only on fibrillogenesis, but also on the mature amyloid fibrils, including stress conditions, in order to avoid undesirable disease progression during chaperone-based therapy.

Probing the allostery in dimeric near-infrared biomarkers derived from the bacterial phytochromes: The impact of the T204A substitution on the inter-monomer interaction.

In dimeric near-infrared (NIR) biomarkers engineered from bacterial phytochromes the covalent binding of BV to the cysteine residue in one monomer of a protein allosterically prevents the chromophore embedded into the pocket of the other monomer from the covalent binding to the cysteine residue. In this work, we analyzed the impact on inter-monomeric allosteric effects in dimeric NIR biomarkers of substitutions at position 204, one of the target residues of mutagenesis at the evolution of these proteins. The T204A substitution in iRFP713, developed on the basis of RpBphP2, and in its mutant variant iRFP713/C15S/V256C, in which the ligand covalent attachment site was changed, resulted in the rearrangement of the hydrogen bond network joining the chromophore with the adjacent amino acids and bound water molecules in its local environment. The change in the intramolecular contacts between the chromophore and its protein environment in iRFP713/C15S/V256C caused by the T204A substitution reduced the negative cooperativity of cofactor binding. We discuss the possible influence of cross-talk between monomers the functioning of full-length phytochromes.

Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore.

Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical "transparency window" of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV.

The unfolding of iRFP713 in a crowded milieu.

The exploring of biological processes in vitro under conditions of macromolecular crowding is a way to achieve an understanding of how these processes occur in vivo. In this work, we study the unfolding of the fluorescent probe iRFP713 in crowded environment in vitro. Previously, we showed that the unfolding of the dimeric iRFP713 is accompanied by the formation of a compact monomer and an intermediate state of the protein. In the intermediate state, the macromolecules of iRFP713 have hydrophobic clusters exposed to the surface of the protein and are prone to aggregation. Concentrated solutions of polyethylene glycol (PEG-8000), Dextran-40 and Dextran-70 with a molecular mass of 8000, 40000 and 70000 Da, respectively, were used to model the conditions for macromolecular crowding. A limited available space provided by all the crowding agents used favors to the enhanced aggregation of iRFP713 in the intermediate state at the concentration of guanidine hydrochloride (GdnHCl), at which the charge of protein surface is neutralized by the guanidine cations. This is in line with the theory of the excluded volume. In concentrated solutions of the crowding agents (240-300 mg/ml), the stabilization of the structure of iRFP713 in the intermediate state is observed. PEG-8000 also enhances the stability of iRFP713 in the monomeric compact state, whereas in concentrated solutions of Dextran-40 and Dextran-70 the resistance of the protein in the monomeric state against GdnHCl-induced unfolding decreases. The obtained data argues for the excluded volume effect being not the only factor that contributes the behavior of biological molecules in a crowded milieu. Crowding agents do not affect the structure of the native dimer of iRFP713, which excludes the direct interactions between the target protein and the crowding agents. PEGs of different molecular mass and Dextran-40/Dextran-70 are known to influence the solvent properties of water. The solvent dipolarity/polarizability and basicity/acidity in aqueous solutions of these crowding agents vary in different ways. The change of the solvent properties in aqueous solutions of crowding agents might impact the functioning of a target protein.

The Pathways of the iRFP713 Unfolding Induced by Different Denaturants.

Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically encoded optical probes for visualization of cellular processes and deep-tissue imaging of cells and organs in living animals. In this work, we show that the steady-state and kinetic dependencies of the various spectral characteristics of iRFP713, developed from the bacterial phytochrome RpBphP2 and recorded at protein unfolding induced by guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GTC), and urea, differ substantially. A study of the unfolding of three single-tryptophan mutant forms of iRFP713 expectedly revealed that protein unfolding begins with the dissociation of the native dimer, while the monomers remain compact. A further increase in the denaturant concentration leads to the formation of an intermediate state of iRFP713 having hydrophobic areas exposed on the protein surface (I). The total surface charge of iRFP713 (pI 5.86) changes from negative to positive with an increase in the concentration of GdnHCl and GTC because the negative charge of glutamic and aspartic acids is neutralized by forming salt bridges between the carboxyl groups and GdnH⁺ ions and because the guanidinium cations bind to amide groups of glutamines and asparagines. The coincidence of both the concentration of the denaturants at which the intermediate state of iRFP713 accumulates and the concentration of GdnH⁺ ions at which the neutralization of the surface charge of the protein in this state is ensured results in strong protein aggregation. This is evidently realized by iRFP713 unfolding by GTC. At the unfolding of the protein by GdnHCl, an intermediate state is populated at higher denaturant concentrations and a strong aggregation is not observed. As expected, protein aggregates are not formed in the presence of the urea. The aggregation of the protein upon neutralization of the charge on the macromolecule surface is the main indicator of the intermediate state of protein. The unfolded state of iRFP713, whose formation is accompanied by a significant decrease in the parameter A, was found to have a different residual structure in the denaturants used.

Near-Infrared Fluorescent Proteins: Multiplexing and Optogenetics across Scales.

Since mammalian tissue is relatively transparent to near-infrared (NIR) light, NIR fluorescent proteins (FPs) engineered from bacterial phytochromes have become widely used probes for non-invasive in vivo imaging. Recently, these genetically encoded NIR probes have been substantially improved, enabling imaging experiments that were not possible previously. Here, we discuss the use of monomeric NIR FPs and NIR biosensors for multiplexed imaging with common visible GFP-based probes and blue light-activatable optogenetic tools. These NIR probes are suitable for visualization of functional activities from molecular to organismal levels. In combination with advanced imaging techniques, such as two-photon microscopy with adaptive optics, photoacoustic tomography and its recent modification reversibly switchable photoacoustic computed tomography, NIR probes allow subcellular resolution at millimeter depths.