Crowded Milieu Research Articles

Flavones Suppress Aggregation and Amyloid Fibril Formation of Human Lysozyme under Macromolecular Crowding Conditions.

The crowded milieu of a biological cell significantly impacts protein aggregation and interactions. Understanding the effects of macromolecular crowding on the aggregation and fibrillation of amyloidogenic proteins is crucial for the treatment of many amyloid-related disorders. Most in vitro studies of protein amyloid formation and its inhibition by small molecules are conducted in dilute buffers, which do not mimic the complexity of the cellular environment. In this study, we used PEGs to simulate macromolecular crowding and examined the inhibitory effects of flavones DHF, baicalein, and luteolin on human lysozyme (HuL) aggregation at pH 2. Naturally occurring flavones have been effective inhibitors of amyloid formation in some proteins. Our findings indicate that while flavones inhibit HuL aggregation and fibrillation in dilute buffer solutions, complete inhibition is observed with a combination of flavones and PEGs, as shown by ThT fluorescence, light scattering, TEM, and AFM studies. The species formed in the presence of PEG 8000 and flavones were less hydrophobic, less toxic, and α-helix-rich compared to control samples, which were hydrophobic and β-sheet-rich, as demonstrated by ANS hydrophobicity, MTT assay, and CD spectroscopy. Fluorescence titration studies of flavones with HuL showed a significant increase in binding constant values under crowding conditions. These findings highlight the importance of macromolecular crowding in modulating protein aggregation and amyloid inhibition. Further studies using disease-causing mutants of HuL and other amyloidogenic proteins are needed to explore the role of macromolecular crowding in small-molecule-mediated modulation and inhibition of protein aggregation and amyloid formation.

Salt Gradient-Induced Phoresis of Vesicles and Enhanced Membrane Fusion in a Crowded Milieu.

Phoresis of biocolloidal objects in response to chemical gradients is a matter of interest among diverse scientific disciplines owing to their importance in the spatiotemporal orchestration of biochemical processes. Although there are reports of soft matter transport/phoresis in the gradient of ions or salts in the aqueous system, their phoretic behavior in the presence of macromolecular crowder is largely unexplored. Notably, cellular cytoplasm is illustrated as a crowded milieu and thereby understanding biomolecular phoresis in the presence of polymeric macromolecules would endorse phoretic behavior in a biomimetic environment. Here, we report the phoresis-induced enhanced aggregation and fusion of vesicles in gradients of monovalent (NaCl) and divalent salt (MgCl2), in the presence of polymeric crowder, polyethylene glycol of molecular weight 400 (PEG 400). Apart from diffusiophoresis, depletion force plays a crucial factor in crowded environments to control localized vesicle aggregation in a salt gradient. This demonstration will potentially show the pathway to future research related to spatiotemporally correlated liposomal transport and membrane-dependent function (such as content mixing and signaling) in a physiologically relevant crowded environment.

Role of Associated Water Dynamics on Protein Stability and Activity in Crowded Milieu.

Macromolecular crowding bridges in vivo and in vitro studies by simulating cellular complexities such as high viscosity and limited space while maintaining the experimental feasibility. Over the last two decades, the impact of macromolecular crowding on protein stability and activity has been a significant topic of study and discussion, though still lacking a thorough mechanistic understanding. This article investigates the role of associated water dynamics on protein stability and activity within crowded environments, using bromelain and Ficoll-70 as the model systems. Traditional crowding theory primarily attributes protein stability to entropic effects (excluded volume) and enthalpic interactions. However, our recent findings suggest that water structure modulation plays a crucial role in a crowded environment. In this report, we strengthen the conclusion of our previous study, i.e., rigid-associated water stabilizes proteins via entropy and destabilizes them via enthalpy, while flexible water has the opposite effect. In the process, we addressed previous shortcomings with a systematic concentration-dependent study using a single-domain protein and component analysis of solvation dynamics. More importantly, we analyze bromelain's hydrolytic activity using the Michaelis-Menten model to understand kinetic parameters like maximum velocity (Vmax) achieved by the system and the Michaelis-Menten coefficient (KM). Results indicate that microviscosity (not the bulk viscosity) controls the enzyme-substrate (ES) complex formation, where an increase in the microviscosity makes the ES complex formation less favorable. On the other hand, flexible associated water dynamics were found to favor the rate of product formation significantly from the ES complex, while rigid associated water hinders it. This study improves our understanding of protein stability and activity in crowded environments, highlighting the critical role of associated water dynamics.

Bridging Soft Interaction and Excluded Volume in Crowded Milieu through Subtle Protein Dynamics.

The impact of macromolecular crowding on biological macromolecules has been elucidated through the excluded volume phenomenon and soft interactions. However, it has often been difficult to provide a clear demarcation between the two regions. Here, using temperature-dependent dynamics (local and global) of the multidomain protein human serum albumin (HSA) in the presence of commonly used synthetic crowders (Dextran 40, PEG 8, Ficoll 70, and Dextran 70), we have shown the presence of a transition that serves as a bridge between the soft and hard regimes. The bridging region is independent of the crowder identity and displays no apparent correlation with the critical overlap concentration of the polymeric crowding agents. Moreover, the dynamics of domains I and II and the protein gating motion respond differently, thereby bringing to the fore the asymmetry underlying the crowder influence on HSA. In addition, solvent-coupled and decoupled protein motions indicate the heterogeneity of the dynamic landscape in the crowded milieu. We also propose an intriguing correlation between protein stability and dynamics, with increased global stability being accompanied by eased local domain motion.

Capturing ultrafast energy flow of a heme protein in crowded milieu

We have investigated the diversity in the effect of crowded milieu on energy flow in the mitochondrial heme protein, cytochrome c.

Intermolecular interactions underlie protein/peptide phase separation irrespective of sequence and structure at crowded milieu

Liquid-liquid phase separation (LLPS) has emerged as a crucial biological phenomenon underlying the sequestration of macromolecules (such as proteins and nucleic acids) into membraneless organelles in cells. Unstructured and intrinsically disordered domains are known to facilitate multivalent interactions driving protein LLPS. We hypothesized that LLPS could be an intrinsic property of proteins/polypeptides but with distinct phase regimes irrespective of their sequence and structure. To examine this, we studied many (a total of 23) proteins/polypeptides with different structures and sequences for LLPS study in the presence and absence of molecular crowder, polyethylene glycol (PEG-8000). We showed that all proteins and even highly charged polypeptides (under study) can undergo liquid condensate formation, however with different phase regimes and intermolecular interactions. We further demonstrated that electrostatic, hydrophobic, and H-bonding or a combination of such intermolecular interactions plays a crucial role in individual protein/peptide LLPS.

Molecular Crowders Can Induce Collapse in Hydrophilic Polymers via Soft Attractive Interactions.

A comprehensive understanding of protein folding and biomolecular self-assembly in the intracellular environment requires obtaining a microscopic view of the crowding effects. The classical view of crowding explains biomolecular collapse in such an environment in terms of the entropic solvent excluded volume effects subjected to hard-core repulsions exerted by the inert crowders, neglecting their soft chemical interactions. In this study, the effects of nonspecific, soft interactions of molecular crowders in regulating the conformational equilibrium of hydrophilic (charged) polymers are examined. Using advanced molecular dynamics simulations, collapse free energies of an uncharged, a negatively charged, and a charge-neutral 32-mer generic polymer are computed. The strength of the polymer-crowder dispersion energy is modulated to examine its effect on polymer collapse. The results show that the crowders preferentially adsorb and drive the collapse of all three polymers. The uncharged polymer collapse is opposed by the change in solute-solvent interaction energy but is overcompensated by the favorable change in the solute-solvent entropy as observed in hydrophobic collapse. However, the negatively charged polymer collapses with a favorable change in solute-solvent interaction energy due to reduction in the dehydration energy penalty as the crowders partition to the polymer interface and shield the charged beads. The collapse of a charge-neutral polymer is opposed by the solute-solvent interaction energy but is overcompensated by the solute-solvent entropy change. However, for the strongly interacting crowders, the overall energetic penalty decreases since the crowders interact with polymer beads via cohesive bridging attractions to induce polymer collapse. These bridging attractions are found to be sensitive to the binding sites of the polymer, since they are absent in the negatively charged or uncharged polymers. These interesting differences in thermodynamic driving forces highlight the crucial role of the chemical nature of the macromolecule as well as of the crowder in determining the conformational equilibria in a crowded milieu. The results emphasize that the chemical interactions of the crowders should be explicitly considered to account for the crowding effects. The findings have implications in understanding the crowding effects on the protein free energy landscapes.

Towards the energy landscape of adenylate kinase in crowded milieu: Activity, conformation, structure and dynamics in sequence

Enzyme function is governed by a complex network of conformational changes and internal dynamics, with the same getting more convoluted in the crowded cellular environment. Here, we have explored an intricate interplay amongst activity, structure, conformation, and dynamics of a multidomain enzyme, AK3L1 (UniProtKB: Q9UIJ7) in the crowded milieu. We have monitored changes in the enzyme landscape in response to the chemical denaturant, urea, under the influence of different concentrations of macromolecular crowders. Extensive experimental analyses using FRET-based domain displacement measurements, sub-nanosecond time scale local dynamics, and global structural changes, along with enzymatic activity studies, have been carried out to get deeper insights into the factors that may modulate the functional landscape of adenylate kinase (AK3L1). It was observed that AK3L1 gets activated at low urea concentrations, whereas higher urea concentrations unfold and thereby deactivate the enzyme. A sequential response of AK3L1 is observed towards external perturbation (urea) occurring through a series of well-defined steps. Incorporation of crowders not only shift the maximum activity of enzyme to a higher urea concentration, but also enhance domain compaction, as revealed by FRET studies. The modulation in enzyme activity and solvation dynamics acting as local response, precede global unfolding of the enzyme, indicating that the structural alterations around the active site are quite decoupled from the large amplitude global transitions.

Associated Water Dynamics Might Be a Key Factor Affecting Protein Stability in the Crowded Milieu.

Over the past 20 years, the most studied and debated aspect of macromolecular crowding is how it affects protein stability. Traditionally, it is explained by a delicate balance between the stabilizing entropic effect and the stabilizing or destabilizing enthalpic effect. However, this traditional crowding theory cannot explain experimental observations like (i) negative entropic effect and (ii) entropy-enthalpy compensation. Herein, we provide experimental evidence that associated water dynamics plays a crucial role in controlling protein stability in the crowded milieu for the first time. We have correlated the modulation of associated water dynamics with the overall stability and its individual components. We showed that rigid associated water would stabilize the protein through entropy but destabilize it through enthalpy. In contrast, flexible associated water destabilizes the protein through entropy but stabilizes through enthalpy. Consideration of entropic and enthalpic modulation through crowder-induced distortion of associated water successfully explains the negative entropic part and entropy-enthalpy compensation. Furthermore, we argued that the relationship between the associated water structure and protein stability should be better understood by individual entropic and enthalpic components instead of the overall stability. Although a huge effort is necessary to generalize the mechanism, this report provides a unique way of understanding the relationship between protein stability and associated water dynamics, which might be a generic phenomenon and should trigger much research in this area.

Mechanism of protein stabilization in crowded solution and preservation in desiccated sugar glass.

The highly crowded cellular milieu includes countless different macromolecules and osmolytes aimed at regulating osmotic stress while protecting proteins and other macromolecules. Of special interest is the formation of cellular glassy matrices composed of sugars, which strongly correlate with the ability of some yeast, nematodes, and tardigrades to survive desiccation and extreme temperature changes. Remarkably, these carbohydrates stabilize proteins in crowded solutions and preserve them in the dry glassy state. Yet, although considerable efforts have been directed to discern how crowding stabilizes proteins, the molecular-level stabilization mechanism has remained elusive. We have followed the effect of various osmolytes on the native state stability of model miniproteins in crowded solutions using circular dichroism (CD) spectroscopy and our newly developed mean-field theory for crowding. We show that protein native state stability and folding thermodynamic signature can be explained using three distinct types of interactions: excluded volume, solvent-osmolyte non-ideal mixing, and osmolyte-protein soft-interactions. Furthermore, using our combination of synchrotron radiation CD spectroscopy together with sampling enhanced molecular dynamics (MD) simulations we have been able to shed light on the stability and new emerging nanostructures of proteins when they are embedded in sugar glass.

Biomolecular Condensates Regulate Enzymatic Activity under a Crowded Milieu: Synchronization of Liquid-Liquid Phase Separation and Enzymatic Transformation.

Cellular crowding plays a key role in regulating the enzymatic reactivity in physiological conditions, which is challenging to realize in the dilute phase. Enzymes drive a wide range of complex metabolic reactions with high efficiency and selectivity under extremely heterogeneous and crowded cellular environments. However, the molecular interpretation behind the enhanced enzymatic reactivity under a crowded milieu is poorly understood. Herein, using the horseradish peroxidase (HRP) and glucose oxidase (GOx) cascade pair, we demonstrate for the first time that macromolecular crowding induces liquid-liquid phase separation (LLPS) via the formation of liquid-like condensates/droplets and thereby increases the intrinsic catalytic efficiencies of HRP and GOx. Both these enzymes undergo crowding induced homotypic LLPS via enthalpically driven multivalent electrostatic as well as hydrophobic interactions. Using a set of kinetic and microscopic experiments, we show that precise synchronization of spontaneous LLPS and enzymatic transformations is key to realize the enhanced enzymatic activity under the crowded environments. Our findings reveal an unprecedented enhancement (91- to 205-fold) in the catalytic efficiency (kcat/Km) of HRP at pH 4.0 within the droplet phase relative to that in the bulk aqueous phase in the presence of different crowders. In addition, we have shown that other enzymes also undergo spontaneous LLPS under macromolecular crowding, signifying the generality of this phenomenon under the crowded environments. More importantly, coalescence driven highly regulated GOx/HRP cascade reactions within the fused droplets have been demonstrated with enhanced activity and specificity under the crowded environments. The present discovery highlights the active role of membraneless condensates in regulating the enzymatic efficacy for complex metabolic reactions under the crowded cellular environments and may find significant importance in the field of biocatalysis.

Molecular crowders modulate ligand binding affinity to G-quadruplex DNA by decelerating ligand association

The study of kinetics of ligand binding to G-quadruplex DNA (GqDNA) structures is of paramount importance for comprehending (possible) ligand-induced anticancer activity involving GqDNA within cells. However, cellular environment is crowded with variety of small- and macro-molecules that occupy ∼30–40 % of cell-volume. While only few earlier studies dealt with deciphering the kinetics of ligands’ interactions with GqDNA in dilute solution, such studies in cell-like crowded milieu are absent. Here we investigate the effect of small and macro molecular crowders, glucose, sucrose and ficoll 70, on the kinetic steps of association and dissociation of a benzophenoxazine-ligand (Cresyl Violet: CV) with human telometic (hybrid) GqDNA structure using fluorescence correlation spectroscopy (FCS), aided by other methods. We find that the binding constants of the ligand to GqDNA change appreciably with nearly fivefold decrease in the presence of ficoll 70, compared to that in pure buffer solution. FCS measurements unfold that this decrease of binding constants is mainly modulated by the viscosity-induced deceleration of the association of the ligand to GqDNA in the crowded solution; however, the rate-determining dissociation rates remain nearly unchanged in the presence of all three crowders. These results have important implications in the context of ligand/GqDNA interactions within cellular environment, which indicate that even if the binding affinity of a ligand to GqDNA structures may be influenced by cellular crowders, they may not influence the unbinding rate of the ligand from a stable ligand/GqDNA complex formed by strong π-π stacking interactions.

Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of β2-microglobulin (β2m). Although high serum β2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigate the effects of human sera on β2m amyloid fibril formation, revealing that sera markedly inhibit amyloid fibril formation. Results from over 100 sera indicate that, although the inhibitory effects of sera deteriorate in long-term dialysis patients, they are ameliorated by maintenance dialysis treatments in the short term. Serum albumin prevents amyloid fibril formation based on macromolecular crowding effects, and decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. We construct a theoretical model assuming cumulative effects of the three risk factors, suggesting the importance of monitoring temporary and accumulated risks to prevent the development of amyloidosis, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu.

The role of Toxoplasma TFIIS-like protein in the early stages of mRNA transcription

BackgroundThe mRNA transcription is a multistep process involving distinct sets of proteins associated with RNA polymerase II (RNAPII) through various stages. Recent studies have highlighted the role of RNAPII-associated proteins in facilitating the assembly of functional complexes in a crowded nuclear milieu. RNAPII dynamics and gene expression regulation have been primarily studied in model eukaryotes like yeasts and mammals and remain largely unchartered in protozoan parasites like Toxoplasma gondii, where considerable gene expression changes accompany stage differentiations. Here we report a key modulator of RNAPII activity, TFIIS in Toxoplasma gondii (TgTFIIS). MethodsA Pull-down assay demonstrated that TgTFIIS binds to RNAPII subunit TgRPB1. Truncation mutants of TFIIS help us define the regions critical for its binding to TgRPB1. Co-immunoprecipitation analysis confirmed the interaction between the native TgTFIIS and TgRPB1. Confocal microscopy revealed a predominantly nuclear localization. Native TgTFIIS was able to bind promoter DNA which was consistent with the CHIP results. ResultsTgTFIIS complements initiation defects in yeast mutants, and the regions implicated in RNAPII binding appeared essential for this function. Interestingly, the C-terminal zinc finger domain necessary for its potential elongation function is dispensable for TgRPB1 binding. TgTFIIS was found to be associated with the promoter region along with its association with the ORF on an RNAPII transcribed gene. ConclusionThe observations were in line with the potential role of TgTFIIS in early events of RNAPII transcription in addition to elongation. General significanceThe study elucidates the potential role of RNAPII-associated proteins in multiple steps of transcription.

Small molecules playing big roles: Tuning material properties of nucleolar condensates

Living cells are enriched with fluidic materials that are full of activity. Biomolecules such as proteins, lipids, and nucleic acids, as well as small regulatory molecules like ATP, need to be organized and regulated in such a crowded milieu to perform cellular functions. Cells achieve this functional coherence by compartmentalizing their constituents into membrane-bound organelles. Intense research over the past decade has revealed that cells can also accomplish intracellular compartmentalization using membrane-less organelles or biomolecular condensates that are formed via phase separation of proteins and nucleic acids (1).

A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor

The electrostatic effects of protein crowding have not been systematically explored. Rather, protein crowding is generally studied with co‐solvents or crowders that are electrostatically neutral, with no methods to measure how the net charge (Z) of a crowder affects protein function. For example, can the activity of an enzyme be affected electrostatically by the net charge of its neighbor in crowded milieu? This paper reports a method for crowding proteins of different net charge to an enzyme via semi‐random chemical crosslinking. As a proof of concept, RNase A was crowded (at distances ≤ the Debye length) via crosslinking to different heme proteins with Z = +8.50 ± 0.04, Z = +6.39 ± 0.12, or Z = −10.30 ± 1.32. Crosslinking did not disrupt the structure of proteins, according to amide H/D exchange, and did not inhibit RNase A activity. For RNase A, we found that the electrostatic environment of each crowded neighbor had significant effects on rates of RNA hydrolysis. Crowding with cationic cytochrome c led to increases in activity, while crowding with anionic “supercharged” cytochrome c or myoglobin diminished activity. Surprisingly, electrostatic crowding effects were amplified at high ionic strength (I = 0.201 M) and attenuated at low ionic strength (I = 0.011 M). This salt dependence might be caused by a unique set of electric double layers at the dimer interspace (maximum distance of 8 Å, which cannot accommodate four layers). This new method of crowding via crosslinking can be used to search for electrostatic effects in protein crowding.

Understanding the nano colloid-protein interaction in crowded milieu

Unique properties of nano colloids make them diverse molecules of biological applications. The antimicrobial activity of nano colloids prepared from Cymbopogon citratus extract has been well documented. However, for the development of nano colloid-based products for human use, in-depth knowledge of the interactions between proteins and nano colloids is essential. Therefore, in this study, the interactions between the nano colloid synthesized using Cymbopogon citrates extract and holo alpha-lactalbumin (h-α-LA) in a crowded milieu was investigated using several techniques. Trp-fluorescence and far-UV CD measurements were carried out under different pH conditions, which showed significant perturbations in the tertiary and alpha-helical and β-sheet structural content of h-α-LA in the presence of nano colloid, P10. Presence of nano colloid in crowded milieu destabilized the protein in terms of midpoint of thermal denaturation (Tm) and Gibbs free energy (ΔGDo). In this manuscript, the effect of nano colloids on protein structure and stability under crowded cellular conditions was investigated. The CcNc in the presence of P10 has significant detrimental effect on protein’s native conformation which leads to decrease in its stability. This study hypothesised that P10 can be used for improving the efficacy of CcNc as antimicrobial agent.

Macromolecular Crowding and Intrinsically Disordered Proteins: A Polymer Physics Perspective

AbstractThe cell is a crowded environment where a relevant fraction of the available space is occupied by proteins, nucleic acids, and metabolites. Here we discuss recent advancements in the understanding of crowding effects on intrinsically disordered proteins. Differently from their structured counterparts, these proteins do not adopt a stable three‐dimensional structure and remain flexible and dynamic in solution. The physics of polymers and colloids provides a framework to interpret how crowding modulates conformations, dynamics, and interactions of disordered proteins. Flory‐Huggins models enable rationalizing the different degree of compaction induced by crowding agents in terms of depletion interactions. The same interactions modulate the diffusion of the disordered proteins in a crowded milieu and the association and dissociation rates when interacting with a ligand. Altogether, this theoretical framework provides new insights into the interpretation of the effects of the cellular environment on disordered proteins.

Genomic-locus tracking reveals the viscoelasticity of chromatin and its local environment in living cells

In eukaryotic cells, genomic DNA is stored and highly packed into chromatin in the crowded milieu of the cell nucleus. Chromatin is highly organized into a hierarchical structure from multiple loops, known as topologically associating domains (TADs), to higher-order structures, compartments. These structures, determining chromatin folding, are dynamic to regulate gene expression through modulating the accessibility of transcription factors and transcriptional machinery. Nucleoplasm, consisting of DNA, RNA, proteins, and small metabolic molecules, is a viscoelastic medium of the nucleus.

A rebinding-assay for measuring extreme kinetics using label-free biosensors

In vitro kinetic measurements allow mechanistic characterization of binding interactions and are particularly valuable throughout drug discovery, from confirmation of on-target binding in early discovery to fine-tuning of drug-binding properties in pre-clinical development. Early chemical matter often exhibits transient kinetics, which remain challenging to measure in a routine drug discovery setting. For example, characterization of irreversible inhibitors has classically relied on the alkylation rate constant, yet this metric fails to resolve its fundamental constituent rate constants, which drive reversible binding kinetics and affinity complex inactivation. In other cases, extremely rapid association processes, which can approach the diffusion limit, also remain challenging to measure. To address these limitations, a practical kinetic rebinding assay is introduced that may be applied for kinetic screening and characterization of compounds. The new capabilities afforded by this probe-based assay emerge from mixed-phase partitioning in a flow-injection configuration and have been implemented using label-free biosensing. A finite element analysis-based biosensor model, simulating inhibition of rebinding within a crowded hydrogel milieu, provided surrogate test data that enabled development and validation of an algebraic model for estimation of kinetic interaction constants. An experimental proof-of-principle demonstrating estimation of the association rate constant, decoupled from the dissociation process, provided further validation.