Effects Of Molecular Crowding Research Articles

Chelate extraction of metal ions in aqueous/chloroform system based on molecular crowding environment

Abstract In this study, we propose a novel concept for the solvent extraction of metal ions (Co, Zn, and Pb) by mimicking a molecular crowding environment using dextran (Dex). The metal ions were extracted from the aqueous phase into the organic phase (chloroform) in the presence of 8-hydroxyquinoline (HQ). The extraction constant of the metal complex (Kex) increased with increasing Dex concentration (CDex) for all metal ions. When examining the dependence of CDex on the four equilibrium constants (distribution coefficient of HQ, acid dissociation of HQ, complexation constant of metal complex (β), and distribution coefficient of the metal complex) that contribute to Kex, only β increased with CDex. This suggests that an increase in, β, a parameter reflecting the molecular crowding effect, results in an increase in Kex. The increase in β was analyzed based on volume exclusion and osmotic pressure effects. The analytical model effectively explained the enhanced the complexation due to the increase in β and volume exclusion, whereas the osmotic pressure suppressed β. Consequently, we unveiled the effect of molecular crowding on the solvent extraction of metal ions for the first time.

Well-Paired-Seq2: High-Throughput and High-Sensitivity Strategy for Characterizing Low RNA-Content Cell/Nucleus Transcriptomes.

Single-cell RNA sequencing (scRNA-seq) is a transformative technology that unravels the intricate cellular state heterogeneity. However, the Poisson-dependent cell capture and low sensitivity in scRNA-seq methods pose challenges for throughput and samples with a low RNA-content. Herein, to address these challenges, we present Well-Paired-Seq2 (WPS2), harnessing size-exclusion and quasi-static hydrodynamics for efficient cell capture. WPS2 exploits molecular crowding effect, tailing activity enhancement in reverse transcription, and homogeneous enzymatic reaction in the initial bead-based amplification to achieve 3116 genes and 8447 transcripts with an average of ∼20000 reads per cell. WPS2 detected 1420 more genes and 4864 more transcripts than our previous Well-Paired-Seq. It sensitively characterizes transcriptomes of low RNA-content single cells and nuclei, overcoming the Poisson limit for cell and barcoded bead capture. WPS2 also profiles transcriptomes from frozen clinical samples, revealing heterogeneous tumor copy number variations and intercellular crosstalk in clear cell renal cell carcinomas. Additionally, we provide the first single-cell-level characterization of rare metanephric adenoma (MA) and uncover potential specific markers. With the advantages of high sensitivity and high throughput, WPS2 holds promise for diverse basic and clinical research.

Molecular crowding electrolytes for stabilizing Zn metal anode in rechargeable aqueous batteries

A solid electrolyte interphase (SEI) with a robust mechanical property and a high ionic conductivity is imperative for high-performance zinc metal batteries. However, it is difficult to form such a SEI directly from an electrolyte. In this work, a molecular crowding effect is based on the introduction of Zn(OTF)2 and Zn(ClO4)2 to 2 mol/L ZnSO4 electrolytes. Simulations and experiments indicate that the Zn(OTF)2 and Zn(ClO4)2 not only create a molecularly crowded electrolyte environment to promote the interaction of Zn2+and OTF−, but also participate in the reduction to construct a robust and high ionic-conductive SEI, thus promoting metal zinc deposition to the (002) crystal surface. With this molecular crowding electrolyte, a high current density of 1 mA/cm2 can be obtained by assembling symmetric batteries with Zn as the anode for over 1000 h. And in a temperature environment of −10 °C, a current density of 1 mA/cm2 can be obtained by assembling symmetric batteries with Zn for over 200 h. Zn//Bi2S3/VS4@C cells achieve a CE rate of up to 99.81% over 1000 cycles. Hence, the utilization of a molecular crowding electrolyte is deemed a highly effective approach to fabricating a sophisticated SEI for a zinc anode.

Valorization of biomass for food protein via deep eutectic solvent extraction: Understanding the extraction mechanism and impact on protein structure and properties

AbstractThe conventional techniques for protein extraction from biomass are not fully aligned with sustainability goals, so it is important to look for some alternate solutions. By simultaneously extracting both soluble and insoluble proteins, deep eutectic solvents (DESs) offer a viable method for valorizing protein‐rich biomass from a variety of sources. Notably, the molecular crowding effects of DESs may have helped unfolded proteins acquire compact and stable conformations, facilitating solubilization and effective extraction. However, there is still a lack of information regarding how DESs interact with proteins and affect the structure and properties of the recovered proteins. To enable their widespread usage as a sustainable method for extracting dietary proteins, the safety of DES‐extracted proteins must also be addressed. In this paper, we review the state of the science in DES‐mediated protein extraction, focusing on the extraction mechanism and the interactions between DESs and proteins. Additionally, important aspects of DES‐mediated protein extraction that could affect the structure, technofunctional, nutritional characteristics, and safety of extracted protein are explored. DES‐based protein extraction could be helpful to valorize different biomasses for the production of food proteins due to the specific features.

Nature-Inspired Molecular-Crowding Enabling Wide-Humidity Range Applicable, Anti-Freezing, and Robust Zwitterionic Hydrogels for On-Skin Electronics.

Hydrogels are currently in the limelight for applications in soft electronics but they suffer from the tendency to lose water or freeze when exposed to dry environments or low temperatures. Molecular crowding is a prevalent occurrence in living cells, in which molecular crowding agents modify the hydrogen bonding structure, causing a significant reduction in water activity. Here, a wide-humidity range applicable, anti-freezing, and robust hydrogel is developed through the incorporation of natural amino acid proline (Pro) and conductive MXene into polyvinyl alcohol (PVA) hydrogel networks. Theoretical calculations reveal that Pro can transform "free water" into "locked water" via the molecular-crowding effect, thereby suppressing water evaporation and ice forming. Accordingly, the prepared hydrogel exhibits high water retention capability, with 77% and 55% being preserved after exposure to 20 °C, 28% relative humidity (RH)and 35 °C, 90% RH for 12h. Meanwhile, Pro lowers the freezing temperature of the hydrogel to 34 °C and enhances its stretchability and strength. Finally, the PVA/Pro/MXene hydrogels are assembled as multifunctional on-skin strain sensors and conductive electrodes to monitor human motions and detect tiny electrophysiological signals. Collectively, this work provides a molecular crowding strategy that will motivate researchers to develop more advanced hydrogels for versatile applications.

Molecular crowding effect in Hantzch pyridine synthesis in polyethylene glycol aqueous solution.

In a molecular crowding environment, the kinetics and thermodynamics differ from those in a diluted solution. Although the molecular crowding effect has been extensively investigated, its fundamental kinetics and thermodynamics remain unclear. In this study, we investigated the change in the rate constant (k) of the Hantzch pyridine reaction in a molecular crowding environment using polyethylene glycol (PEG). While the k value increased to a PEG concentration (CPEG) of 10 vol%, a decreasing trend was observed for CPEG > 20 vol%. This intriguing behavior was analyzed based on the increase in reactant activity due to volume exclusion and the decrease in water activity due to osmotic pressure. Volume exclusion and osmotic pressure had opposing effects on the reaction, which were positive for volume exclusion and negative for osmotic pressure. We found that k decreased when the negative effect of the osmotic pressure surpassed the volume exclusion effect.

Insights into In Vivo Environmental Effects on Quantitative Biochemistry in Single Cells.

Biomacromolecules exist and function in a crowded and spatially confined intracellular milieu. Single-cell analysis has been an essential tool for deciphering the molecular mechanisms of cell biology and cellular heterogeneity. However, a sound understanding of in vivo environmental effects on single-cell quantification has not been well established. In this study, via cell mimicking with giant unilamellar vesicles and single-cell analysis by an approach called plasmonic immunosandwich assay (PISA) that we developed previously, we investigated the effects of two in vivo environmental factors, i.e., molecular crowding and spatial confinement, on quantitative biochemistry in the cytoplasm of single cells. We find that molecular crowding greatly affects the biomolecular interactions and immunorecognition-based detection while the effect of spatial confinement in cell-sized space is negligible. Without considering the effect of molecular crowding, the results by PISA were found to be apparently under-quantitated, being only 29.5-50.0% of those by the calibration curve considering the effect of molecular crowding. We further demonstrated that the use of a calibration curve established with standard solutions containing 20% (wt) polyethylene glycol 6000 can well offset the effect of intracellular crowding and thereby provide a simple but accurate calibration for the PISA measurement. Thus, this study not only sheds light on how intracellular environmental factors influence biomolecular interactions and immunorecognition-based single-cell quantification but also provides a simple but effective strategy to make the single-cell analysis more accurate.

Insights into the Mechanism of Tryptophan Fluorescence Quenching due to Synthetic Crowding Agents: A Combined Experimental and Computational Study.

Intrinsic tryptophan fluorescence spectroscopy is an important tool for examining the effects of molecular crowding and confinement on the structure, dynamics, and function of proteins. Synthetic crowders such as dextran, ficoll, polyethylene glycols, polyvinylpyrrolidone, and their respective monomers are used to mimic crowded intracellular environments. Interactions of these synthetic crowders with tryptophan and the subsequent impact on its fluorescence properties are therefore critically important for understanding the possible interference created by these crowders. In the present study, the effects of polymer and monomer crowders on tryptophan fluorescence were assessed by using experimental and computational approaches. The results of this study demonstrated that both polymer and monomer crowders have an impact on the tryptophan fluorescence intensity; however, the molecular mechanisms of quenching were different. Using Stern-Volmer plots and a temperature variation study, a physical basis for the quenching mechanism of commonly used synthetic crowders was established. The quenching of free tryptophan was found to involve static, dynamic, and sphere-of-action mechanisms. In parallel, computational studies employing Kohn-Sham density functional theory provided a deeper insight into the effects of intermolecular interactions and solvation, resulting in differing quenching modes for these crowders. Taken together, the study offers new physical insights into the quenching mechanisms of some commonly used monomer and polymer synthetic crowders.

Contrasting effects of molecular crowding on the membrane-perturbing and chaperone-like activities of major bovine seminal plasma protein, PDC-109

Crowded environments inside cells and biological fluids greatly affect protein stability and activity. PDC-109, a polydisperse oligomeric protein of the bovine seminal plasma selectively binds choline phospholipids on the sperm cell surface and causes membrane destabilization and lipid efflux, leading to acrosome reaction. PDC-109 also exhibits chaperone-like activity (CLA) and protects client proteins against various kinds of stress, such as high temperature and low pH. In the present work, we have investigated the effect of molecular crowding on these two different activities of PDC-109 employing Dextran 70 (D70) – a widely used polymeric dextran – as the crowding agent. The results obtained show that presence of D70 markedly increases membrane destabilization by PDC-109. Isothermal titration calorimetric studies revealed that under crowded condition the binding affinity of PDC-109 for choline phospholipids increases approximately 3-fold, which could in turn facilitate membrane destabilization. In contrast, under identical conditions, its CLA was reduced significantly. The decreased CLA could be correlated to reduced surface hydrophobicity, which was due to stabilization of the protein oligomers. These results establish that molecular crowding exhibits contrasting effects on two different functional activities of PDC-109 and highlight the importance of microenvironment of proteins in modulating their functional activities.

Leveraging liquid-liquid phase separation and volume modulation to regulate the enzymatic activity of formate dehydrogenase

Engineering of reaction media is an exciting alternative for modulating kinetic properties of biocatalytic reactions. We addressed the combined effect of an aqueous two-phase system (ATPS) and high hydrostatic pressure on the kinetics of the Candida boidinii formate dehydrogenase-catalyzed oxidation of formate to CO2. Pressurization was found to lead to an increase of the binding affinity (decrease of KM, respectively) and a decrease of the turnover number, kcat. The experimental approach was supported using thermodynamic modeling with the electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) equation of state to predict the liquid-liquid phase separation and the molecular crowding effect of the ATPS on the kinetic properties. The ePC-SAFT was able to quantitatively predict the KM-values of the substrate in both phases at 1 bar as well as up to a pressure of 1000 bar. The framework presented enables significant advances in bioprocess engineering, including the design of processes with significantly fewer experiments and trial-and-error approaches.

In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics.

In cells, the formation of RNA/DNA hybrid duplexes regulates gene expression and modification. The environment inside cellular organelles is heterogeneously crowded with high concentrations of biomolecules that affect the structure and stability of RNA/DNA hybrid duplexes. However, the detailed environmental effects remain unclear. Therefore, the mechanistic details of the effect of such molecular crowding were investigated at the molecular level by using thermodynamic and nuclear magnetic resonance analyses, revealing structure-dependent destabilization of the duplexes under crowded conditions. The transition from B- to A-like hybrid duplexes due to a change in conformation of the DNA strand guided by purine-pyrimidine asymmetry significantly increased the hydration number, which resulted in greater destabilization by the addition of cosolutes. By quantifying the individual contributions of environmental factors and the bulk structure of the duplex, we developed a set of parameters that predict the stability of hybrid duplexes with conformational dissimilarities under diverse crowding conditions. A comparison of the effects of environmental conditions in living cells and in vitro crowded solutions on hybrid duplex formation using the Förster resonance energy transfer technique established the applicability of our parameters to living cells. Moreover, our derived parameters can be used to estimate the efficiency of transcriptional inhibition, genome editing, and silencing techniques in cells. This supports the usefulness of our parameters for the visualization of cellular mechanisms of gene expression and the development of nucleic acid-based therapeutics targeting different cells.

DNA Nanomachine-Driven Heterogeneous Quadratic Amplification for Sensitive and Programmable miRNA Profiling.

Inspired by the molecular crowding effect in biological systems, a novel heterogeneous quadratic amplification molecular circuit (HEQAC) was developed for sensitive bimodal miRNA profiling (HEQAC-BMP) by combining an MNAzyme-based DNA nanomachine with an entropy-driven catalytic hairpin assembly (E-CHA) autocatalytic circuit. Utilizing ferromagnetic nanomaterials as the substrate for DNA nanomachines, a biomimetic heterogeneous interface was established; thus, a localized molecular crowding system was created that can elevate the local reaction concentration and accelerate the molecular recognition process for a significant threshold signal. Simultaneously, the threshold signal undergoes further amplification by E-CHA and is transformed into a chemical signal, enabling a colorimetric-fluorescence bimodal signal readout. The HEQAC-BMP enables miRNA detection from 10 aM to 10 nM with detection limits of 3.7 aM (colorimetry) and 4.8 aM (fluorometry), respectively. Moreover, the design principle and strategy of HEQAC-BMP can be customized to address other critical viruses or diseases with life-threatening and socioeconomic impacts, enhancing healthcare outcomes for individuals.

Tuning Hydrophobicity of Paper Substrates for Effective Colorimetricdetection of Glucose and Nucleic acids.

This study investigated the colorimetric response of standard glucose, serum glucose, and nucleic acid assays on various paper surfaces with different wettability, including hydrophilic, hydrophobic, and nearly superhydrophobic surfaces. Water contact angles (WCA) formed by water droplets on each surface were measured using ImageJ software. The hydrophilic surface showed no contact angle, while the hydrophobic and nearly superhydrophobic surfaces exhibited contact angles of 115.667° and 133.933°, respectively. The colorimetric sensitivity of the standard glucose assay was analyzed on these surfaces, revealing enhanced sensitivity on the nearly superhydrophobic surface due to the high molecular crowding effect owing to its non-wetting behavior and eventually confined reaction product at the sample loading zone. The hydrophobic nature of the surface restricts the spreading and diffusion of the reaction product, leading to a controlled and localized concentration of the assay product leading to moderate colorimetric intensity. On the other hand, the hydrophilic surface showed the least enhancement in colorimetric sensitivity; this is attributed to the high wettability of the hydrophilic surface causing the reaction product to spread extensively, resulting in a larger area of dispersion and consequently a lower colorimetric intensity. The measured limit of detection (LOD) for nucleic acid on nearly superhydrophobic surfaces was found to be 16.15ng/µL, which was almost four-fold lower than on hydrophilic surfaces (60.08ng/µL). Additionally, the LODs of standard glucose and clinical serum samples were two-fold lower on nearly superhydrophobic surfaces compared to hydrophilic surfaces. Our findings clearly highlight the promising potential of utilizing superhydrophobic surfaces to significantly enhance colorimetric sensitivity in paper-based diagnostic applications. This innovative approach holds promise for advancing point-of-care diagnostics and improving disease detection in resource-limited settings.

Molecular crowding promotes the aggregation of parallel structured G-quadruplexes

G-quadruplexes are widely distributed in cells and are usually essential in mediating biological processes. The intracellular environment is often in a state of molecular crowding, and the current research considerably focuses on the effect of molecular crowding on the conformation of telomeric G-quadruplexes. However, G-quadruplex-forming oligonucleotides are primarily located in the promoter region of the proto-oncogene and on mRNA inside the cell and are reported to fold into parallel structures. Thus, studying the interaction mechanism between ligands and parallel structured G-quadruplexes under crowding conditions is crucial for the design of drugs targeting G-quadruplexes. In our study, molecular crowding was simulated through polyethylene glycol with an average molecular weight of 200 (PEG200) to investigate the parallel structure of the canonical G-quadruplexes c-KIT1, c-MYC, and 32KRAS and their interactions with ligands. Circular dichroism (CD) spectral scanning, fluorescence resonance energy transfer (FRET), and native polyacrylamide gel electrophoresis (PAGE) analysis revealed that molecular crowding failed to induce oligonucleotides to form parallel G-quadruplex structures in the explored model sequences while induced telomeric G-rich sequences to form antiparallel G-quadruplexes in solution without K+. Molecular crowding did not induce changes in their parallel structures but promoted the formation of G-quadruplex aggregates. Moreover, to some extent, molecular crowding also induced a looser structure of the monomer G-quadruplexes. Further studies showed that molecular crowding did not alter the binding stoichiometry of the ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate (RHPS4) to c-KIT1, while it inhibited its interaction with parallel structured G-quadruplexes. This work provides new insights into developing anticancer drugs targeting parallel structured G-quadruplexes.

Effects of Molecular Crowding on the Structure, Stability, and Interaction with Ligands of G-quadruplexes.

G-quadruplexes (G4s) are widely found in cells and have significant biological functions, which makes them a target for screening antitumor and antiviral drugs. Most of the previous research on G4s has been conducted mainly in diluted solutions. However, cells are filled with organelles and many biomolecules, resulting in a constant state of a crowded molecular environment. The conformation and stability of some G4s were found to change significantly in the molecularly crowded environment, and interactions with ligands were disturbed to some extent. The structure of the G4s and their biological functions are correlated, and the effect of the molecularly crowded environment on G4 conformational transitions and interactions with ligands should be considered in drug design targeting G4s. This review discusses the changes in the conformation and stability of G4s in a physiological environment. Moreover, the mechanism of action of the molecularly crowded environment affecting the G4 has been further reviewed based on previous studies. Furthermore, current challenges and future research directions are put forward. This review has implications for the design of drugs targeting G4s.

WNK1, a molecular crowding sensor, links phase separation to cellular physiological stress.

WNK1, a molecular crowding sensor, links phase separation to cellular physiological stress.

Precipitation-free aluminum-air batteries with high capacity and durable service life

Aluminum-air batteries are potential candidates for future large-scale energy storage/conversion due to their high safety and energy density. However, aluminum-air batteries face the challenges of continuously accumulated discharge by-products and undesired parasitic hydrogen evolution reaction (HER), which induce inferior performance and service life. Here, HER-suppressing and precipitation-free molecular crowding electrolytes are developed using sodium polyacrylate (PANa) as the crowding agent by taking advantage of its molecular crowding effect and scale inhibition property. The introduced PANa reconfigures the hydrogen-bond networks of electrolyte to suppress the HER by molecular crowding effect. A more uniform electric field distribution can be found on the surface of aluminum anode in the electrolyte containing PANa by employing Comsol multiphysics simulation. In addition, the anion long chains of PANa are electrostatically adsorbed on the surface of Al(OH)3 by-products micro-crystal, resulting in the same negative charge on the surface of the micro-crystal to prevent further aggregation and precipitation. The fabricated flow-based aluminum-air battery exhibits an outstanding specific capacity of 2096 mAh g−1, demonstrating the remarkable positive effect of PANa-based molecular crowding electrolyte in aluminum-air batteries. This work provides new light on aqueous electrolyte design for high capacity and precipitation-free aluminum-air batteries.

Thermodynamic complexation mechanism of zinc ion with 8-hydroxyquinoline-5-sulfonic acid in molecular crowding environment

A crowded molecular environment, which is universally observed in biological cells, changes the thermodynamics and kinetics of biochemical reactions compared to bulk solution. Although molecular crowding has received significant attention, a fundamental explanation of the molecular crowding effect based on thermodynamic parameters, including enthalpy (ΔH) and entropy changes (ΔS), is insufficient. Herein, the complexation mechanism of zinc ions with quinolinol derivative in a molecular crowding environment was investigated using polyethylene glycol (PEG) based on the changes in ΔH and ΔS. For 1:1 complexation, which is only promoted by the osmotic pressure effect, ΔH and ΔS decreased with increasing PEG concentration (CPEG) because the number of dehydration molecules decreased. In the 1:2 complexation system, which is affected by the osmotic pressure and volume exclusion effects, no obvious dependences of ΔH and ΔS on CPEG were observed. This is because the osmotic pressure and volume exclusion effects contributed oppositely to ΔH and ΔS. The results presented herein provide fundamental knowledge for protein-metal interactions, and it is expected that these data will attract the attention of many physical chemists and biochemists.

Effects of Molecular Crowding and Betaine on HSPB5 Interactions, with Target Proteins Differing in the Quaternary Structure and Aggregation Mechanism

The aggregation of intracellular proteins may be enhanced under stress. The expression of heat-shock proteins (HSPs) and the accumulation of osmolytes are among the cellular protective mechanisms in these conditions. In addition, one should remember that the cell environment is highly crowded. The antiaggregation activity of HSPB5 and the effect on it of either a crowding agent (polyethylene glycol (PEG)) or an osmolyte (betaine), or their mixture, were tested on the aggregation of two target proteins that differ in the order of aggregation with respect to the protein: thermal aggregation of glutamate dehydrogenase and DTT-induced aggregation of lysozyme. The kinetic analysis of the dynamic light-scattering data indicates that crowding can decrease the chaperone-like activity of HSPB5. Nonetheless, the analytical ultracentrifugation shows the protective effect of HSPB5, which retains protein aggregates in a soluble state. Overall, various additives may either improve or impair the antiaggregation activity of HSPB5 against different protein targets. The mixed crowding arising from the presence of PEG and 1 M betaine demonstrates an extraordinary effect on the oligomeric state of protein aggregates. The shift in the equilibrium of HSPB5 dynamic ensembles allows for the regulation of its antiaggregation activity. Crowding can modulate HSPB5 activity by affecting protein-protein interactions.

Simultaneous regulation on electrolyte structure and electrode interface with glucose additive for high-energy aluminum metal-air batteries

Aqueous metal-based batteries have attracted great attention due to their high energy density and high safety. However, currently suppressing the severe parasitic hydrogen evolution reaction (HER) at the metal anodes in aqueous electrolytes remains challenging. Here, a novel “two birds with one stone” strategy of simultaneously reducing water activity and constructing a protective layer on Al anode with a green and low-cost glucose additive is proposed to suppress the HER. Theoretical studies and experimental characterizations confirm that the glucose additive could reduce water activity via the “molecular crowding effect” and form an organic protective layer on Al metal via chemical adsorption. The dual-functional glucose additive exhibits significant suppression on the HER and self-discharge of Al-metal anodes, which endows a flow-based Al-air battery with ultra-high specific capacity of 2886.7 mAh g Al −1 and energy density of 3675.1 Wh kg Al −1 . This study not only blazes a new path for suppressing HER on Al-metal anodes but also casts new light on aqueous electrolyte design and the stabilization of metal anodes.