Folding of poly-amino acids and intrinsically disordered proteins in overcrowded milieu induced by pH change

pH-induced structural changes of the synthetic homopolypeptides poly-E, poly-K, poly-R, and intrinsically disordered proteins (IDPs) prothymosin alpha (ProT alpha) and linker histone H1, in concentrated PEG solutions simulating macromolecular crowding conditions within the membrane-less organelles, were characterized. The conformational transitions of the studied poly-amino acids in the concentrated PEG solutions depend on the polymerization degree of these homopolypeptides, the size of their side chains, the charge distribution of the side chains, and the crowding agent concentration. The results obtained for poly-amino acids are valid for IDPs having a significant total charge. The overcrowded conditions promote a significant increase in the cooperativity of the pH-induced coil-alpha-helix transition of ProTa and provoke histone H1 aggregation. The most favorable conditions for the pH-induced structural transitions in concentrated PEG solutions are realized when the charged residues are grouped in blocks, and when the distance between the end of the side group carrying charge and the backbone is small. Therefore, the block-wise distribution of charged residues within the IDPs not only plays an important role in the liquid-liquid phase transitions, but may also define the expressivity of structural transitions of these proteins in the overcrowded conditions of the membrane-less organelles. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe

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

Protein-Ligand Interactions of the D-Galactose/D-Glucose-Binding Protein as a Potential Sensing Probe of Glucose Biosensors

Abstract. In this paper we have studied peculiarities of protein-ligand interaction under different conditions. We have shown that guanidine hydrochloride (GdnHCI) unfolding-refolding of GGBP in the presence of glucose (Glc) is reversible, but the equilibrium curves of complex refolding-unfolding have been attained only after 10-day incubation of GGBP/Glc in the presence of GdnHCl. This effect has not been revealed at heat-induced GGBP/Glc denaturation. Slow equilibration between the native protein in GGBP/Glc complex and the unfolded state of protein in the GdnHCl presence is connected with increased viscosity of solution at moderate and high GdnHCl concentrations which interferes with diffusion of glucose molecules. Thus, the limiting step of the unfolding-refolding process of the complex GGBP/Glc is the disruption/tuning of the configuration fit between the protein in the native state and the ligand

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 Role of Liquid–Liquid Phase Separation in Actin Polymerization

To date, it has been shown that the phenomenon of liquid–liquid phase separation (LLPS) underlies many seemingly completely different cellular processes. This provided a new idea of the spatiotemporal organization of the cell. The new paradigm makes it possible to provide answers to many long-standing, but still unresolved questions facing the researcher. In particular, spatiotemporal regulation of the assembly/disassembly of the cytoskeleton, including the formation of actin filaments, becomes clearer. To date, it has been shown that coacervates of actin-binding proteins that arise during the phase separation of the liquid–liquid type can integrate G-actin and thereby increase its concentration to initiate polymerization. It has also been shown that the activity intensification of actin-binding proteins that control actin polymerization, such as N-WASP and Arp2/3, can be caused by their integration into liquid droplet coacervates formed by signaling proteins on the inner side of the cell membrane

Multi-functionality of Proteins Involved in GPCR and G Protein Signaling: Making Sense of Structure–function Continuum with Intrinsic Disorder-based Proteoforms

GPCR–G protein signaling system recognizes a multitude of extracellular ligands and triggers a variety of intracellular signaling cascades in response. In humans, this system includes more than 800 various GPCRs and a large set of heterotrimeric G proteins. Complexity of this system goes far beyond a multitude of pair-wise ligand–GPCR and GPCR–G protein interactions. In fact, one GPCR can recognize more than one extracellular signal and interact with more than one G protein. Furthermore, one ligand can activate more than one GPCR, and multiple GPCRs can couple to the same G protein. This defines an intricate multifunctionality of this important signaling system. Here, we show that the multifunctionality of GPCR–G protein system represents an illustrative example of the protein structure–function continuum, where structures of the involved proteins represent a complex mosaic of differently folded regions (foldons, non-foldons, unfoldons, semi-foldons, and inducible foldons). The functionality of resulting highly dynamic conformational ensembles is fine-tuned by various post-translational modifications and alternative splicing, and such ensembles can undergo dramatic changes at interaction with their specific partners. In other words, GPCRs and G proteins exist as sets of conformational/basic, inducible/modified, and functioning proteoforms characterized by a broad spectrum of structural features and possessing various functional potentials

Stress-Induced Membraneless Organelles in Eukaryotes and Prokaryotes: Bird’s-Eye View

Stress is an inevitable part of life. An organism is exposed to multiple stresses and overcomes their negative consequences throughout its entire existence. A correlation was established between life expectancy and resistance to stress, suggesting a relationship between aging and the ability to respond to external adverse effects as well as quickly restore the normal regulation of biological processes. To combat stress, cells developed multiple pro-survival mechanisms, one of them is the assembly of special stress-induced membraneless organelles (MLOs). MLOs are formations that do not possess a lipid membrane but rather form as a result of the “liquid–liquid” phase separation (LLPS) of biopolymers. Stress-responsive MLOs were found in eukaryotes and prokaryotes, they form as a reaction to the acute environmental conditions and are dismantled after its termination. These compartments function to prevent damage to the genetic and protein material of the cell during stress. In this review, we discuss the characteristics of stress-induced MLO-like structures in eukaryotic and prokaryotic cells

The Role of Liquid–Liquid Phase Separation in Actin Polymerization

To date, it has been shown that the phenomenon of liquid–liquid phase separation (LLPS) underlies many seemingly completely different cellular processes. This provided a new idea of the spatiotemporal organization of the cell. The new paradigm makes it possible to provide answers to many long-standing, but still unresolved questions facing the researcher. In particular, spatiotemporal regulation of the assembly/disassembly of the cytoskeleton, including the formation of actin filaments, becomes clearer. To date, it has been shown that coacervates of actin-binding proteins that arise during the phase separation of the liquid–liquid type can integrate G-actin and thereby increase its concentration to initiate polymerization. It has also been shown that the activity intensification of actin-binding proteins that control actin polymerization, such as N-WASP and Arp2/3, can be caused by their integration into liquid droplet coacervates formed by signaling proteins on the inner side of the cell membrane

Intrinsically Disordered Proteins in Crowded Milieu: When Chaos Prevails Within the Cellular Gumbo

Effects of macromolecular crowding on structural and functional properties of ordered proteins, their folding, interactability, and aggregation are well documented. Much less is known about how macromolecular crowding might affect structural and functional behaviour of intrinsically disordered proteins (IDPs) or intrinsically disordered protein regions (IDPRs). To fill this gap, this review represents a systematic analysis of the available literature data on the behaviour of IDPs/IDPRs in crowded environment. Although it was hypothesized that, due to the excluded-volume effects present in crowded environments, IDPs/IDPRs would invariantly fold in the presence of high concentrations of crowding agents or in the crowded cellular environment, accumulated data indicate that, based on their response to the presence of crowders, IDPs/IDPRs can be grouped into three major categories, foldable, non-foldable, and unfoldable. This is because natural cellular environment is not simply characterized by the presence of high concentration of “inert” macromolecules, but represents an active milieu, components of which are engaged in direct physical interactions and soft interactions with target proteins. Some of these interactions with cellular components can cause (local) unfolding of query proteins. In other words, since crowding can cause both folding and unfolding of an IDP or its regions, the outputs of the placing of a query protein to the crowded environment would depend on the balance between these two processes. As a result, and because of the spatio-temporal heterogeneity in structural organization of IDPs, macromolecular crowding can differently affect structures of different IDPs. Recent studies indicate that some IDPs are able to undergo liquid–liquid-phase transitions leading to the formation of various proteinaceous membrane-less organelles (PMLOs). Although interiors of such PMLOs are self-crowded, being characterized by locally increased concentrations of phase-separating IDPs, these IDPs are minimally foldable or even non-foldable at all (at least within the physiologically safe time-frame of normal PMLO existence)

On the Role of Normal Aging Processes in the Onset and Pathogenesis of Diseases Associated with the Abnormal Accumulation of Protein Aggregates

Aging is a prime systemic cause of various age-related diseases, in particular, proteinopathies. In fact, most diseases associated with protein misfolding are sporadic, and their incidence increases with aging. This review examines the process of protein aggregate formation, the toxicity of such aggregates, the organization of cellular systems involved in proteostasis, and the impact of protein aggregates on important cellular processes leading to proteinopathies. We also analyze how manifestations of aging (mitochondrial dysfunction, dysfunction of signaling systems, changes in the genome and epigenome) facilitate pathogenesis of various proteinopathies either directly, by increasing the propensity of key proteins for aggregation, or indirectly, through dysregulation of stress responses. Such analysis might help in outlining approaches for treating proteinopathies and extending healthy longevity