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International Journal of Molecular... Jan 2021Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation,...
Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen-NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.
Topics: Biosensing Techniques; Genes, Reporter; HEK293 Cells; Humans; Intracellular Space; Luciferases; Luminescent Measurements; Nanotechnology; Osmolar Concentration; Recombinant Proteins; Structure-Activity Relationship
PubMed: 33445497
DOI: 10.3390/ijms22020677 -
Biochemistry Oct 2022Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those...
Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those imposed by post-translational modifications, impacts chromatin dynamics, protein binding, and the fate of DNA damage. There is evidence that N-terminal histone tails affect the local ionic environment within a nucleosome core particle (NCP), but this phenomenon is not well understood. Determining the modulation of the local ionic environment within an NCP by histone tails could help uncover the underlying mechanisms of their functions and effects. Utilizing bottom-up syntheses of NCPs containing wild-type or mutated histones and a fluorescent probe that is sensitive to the local ionic environment, we show that interaction with positively charged N-terminal tails increases the local ionic strength near nucleosomal DNA. The effect is diminished by replacing positively charged residues with neutral ones or deleting a tail in its entirety. Replacing the fluorescent probe with the major DNA methylation product, 7-methyl-2'-deoxyguanosine (MdG), revealed changes in the depurination rate constant varying inversely with local ionic strength. These data indicate that the MdG hydrolysis rates depend on and also inform on local ionic strength in an NCP. Overall, histone tail charge contributes to the complexity of the NCP structure and function by modulating the local ionic strength.
Topics: Chromatin; DNA; Deoxyguanosine; Fluorescent Dyes; Histones; Nucleosomes; Osmolar Concentration
PubMed: 36136907
DOI: 10.1021/acs.biochem.2c00342 -
International Journal of Molecular... Aug 2022One of the commonly accepted approaches to estimate protein-protein interactions (PPI) in aqueous solutions is the analysis of their translational diffusion. The present... (Review)
Review
One of the commonly accepted approaches to estimate protein-protein interactions (PPI) in aqueous solutions is the analysis of their translational diffusion. The present review article observes a phenomenological approach to analyze PPI effects via concentration dependencies of self- and collective translational diffusion coefficient for several spheroidal proteins derived from the pulsed field gradient NMR (PFG NMR) and dynamic light scattering (DLS), respectively. These proteins are rigid globular α-chymotrypsin (ChTr) and human serum albumin (HSA), and partly disordered α-casein (α-CN) and β-lactoglobulin (β-Lg). The PPI analysis enabled us to reveal the dominance of intermolecular repulsion at low ionic strength of solution (0.003-0.01 M) for all studied proteins. The increase in the ionic strength to 0.1-1.0 M leads to the screening of protein charges, resulting in the decrease of the protein electrostatic potential. The increase of the van der Waals potential for ChTr and α-CN characterizes their propensity towards unstable weak attractive interactions. The decrease of van der Waals interactions for β-Lg is probably associated with the formation of stable oligomers by this protein. The PPI, estimated with the help of interaction potential and idealized spherical molecular geometry, are in good agreement with experimental data.
Topics: Biophysical Phenomena; Caseins; Diffusion; Humans; Osmolar Concentration; Protein Processing, Post-Translational; Static Electricity
PubMed: 36012504
DOI: 10.3390/ijms23169240 -
Biophysical Journal Apr 2021Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions,...
Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and NaSO) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the acceptor (mCitrine) in KE (lysine-glutamate), arginine-aspartate, and arginine-glutamate ionic strength sensors with variable amino acid sequences. Similar experiments were carried out on the cleaved sensors as well as an E6G2 construct, which has neutral α-helices in the linker region, as a control. Our results show distinct dynamics of the intact and cleaved sensors. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases. Our chemical equilibrium analyses of the collapsed-to-stretched conformational state transition of KE reveal that the corresponding equilibrium constant and standard Gibbs free energy changes are ionic strength dependent. We also tested the existing theoretical models for FRET analyses using steady-state anisotropy, which reveal that the angle between the dipole moments of the donor and acceptor in the KE sensor are sensitive to the ionic strength. These results help establish the time-resolved depolarization dynamics of these genetically encoded donor-acceptor pairs as a quantitative means for FRET analysis, which complement traditional methods such as time-resolved fluorescence for future in vivo studies.
Topics: Anisotropy; Biosensing Techniques; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Osmolar Concentration
PubMed: 33582140
DOI: 10.1016/j.bpj.2021.01.035 -
The Journal of Physical Chemistry. B Nov 2020Hydrophobicity is a phenomenon of great importance in biology, chemistry, and biochemistry. It is defined as the interaction between nonpolar molecules or groups in...
Hydrophobicity is a phenomenon of great importance in biology, chemistry, and biochemistry. It is defined as the interaction between nonpolar molecules or groups in water and their low solubility. Hydrophobic interactions affect many processes in water, for example, complexation, surfactant aggregation, and coagulation. These interactions play a pivotal role in the formation and stability of proteins or biological membranes. In the present study, we assessed the effect of ionic strength, solute size, and shape on hydrophobic interactions between pairs of nonpolar particles. Pairs of methane, neopentane, adamantane, fullerene, ethane, propane, butane, hexane, octane, and decane were simulated by molecular dynamics in AMBER 16.0 force field. As a solvent, TIP3P and TIP4PEW water models were used. Potential of mean force (PMF) plots of these dimers were determined at four values of ionic strength, 0, 0.04, 0.08, and 0.40 mol/dm, to observe its impact on hydrophobic interactions. The characteristic shape of PMFs with three extrema (contact minimum, solvent-separated minimum, and desolvation maximum) was observed for most of the compounds for hydrophobic interactions. Ionic strength affected hydrophobic interactions. We observed a tendency to deepen contact minima with an increase in ionic strength value in the case of spherical and spheroidal molecules. Additionally, two-dimensional distribution functions describing water density and average number of hydrogen bonds between water molecules were calculated in both water models for adamantane and hexane. It was observed that the density of water did not significantly change with the increase in ionic strength, but the average number of hydrogen bonds changed. The latter tendency strongly depends on the water model used for simulations.
Topics: Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Osmolar Concentration; Solutions; Water
PubMed: 33147018
DOI: 10.1021/acs.jpcb.0c06399 -
Polymorphism of Alpha-Synuclein Amyloid Fibrils Depends on Ionic Strength and Protein Concentration.International Journal of Molecular... Nov 2021Protein aggregate formation is linked with multiple amyloidoses, including Alzheimer's and Parkinson's diseases. Currently, the understanding of such fibrillar structure...
Protein aggregate formation is linked with multiple amyloidoses, including Alzheimer's and Parkinson's diseases. Currently, the understanding of such fibrillar structure formation and propagation is still not sufficient, the outcome of which is a lack of potent, anti-amyloid drugs. The environmental conditions used during in vitro protein aggregation assays play an important role in determining both the aggregation kinetic parameters, as well as resulting fibril structure. In the case of alpha-synuclein, ionic strength has been shown as a crucial factor in its amyloid aggregation. In this work, we examine a large sample size of alpha-synuclein aggregation reactions under thirty different ionic strength and protein concentration combinations and determine the resulting fibril structural variations using their dye-binding properties, secondary structure and morphology. We show that both ionic strength and protein concentration determine the structural variability of alpha-synuclein amyloid fibrils and that sometimes even identical conditions can result in up to four distinct types of aggregates.
Topics: Amyloid; In Vitro Techniques; Kinetics; Osmolar Concentration; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Structure, Secondary; alpha-Synuclein
PubMed: 34830264
DOI: 10.3390/ijms222212382 -
The Journal of Physical Chemistry. B Mar 2023Single-molecule DNA studies have improved our understanding of the DNAs' structure and their interactions with other molecules. A variety of DNA labeling dyes are...
Single-molecule DNA studies have improved our understanding of the DNAs' structure and their interactions with other molecules. A variety of DNA labeling dyes are available for single-molecule studies, among which the bis-intercalating dye YOYO-1 and mono-intercalating dye YO-PRO-1 are widely used. They have an extraordinarily strong affinity toward DNA and are bright with a high quantum yield (>0.5) when bound to DNAs. However, it is still not clear how these dyes behave in DNA molecules under higher ionic strength and strong buffer flow. Here, we have studied the effect of ionic strength and flow rate of buffer on their binding in single DNA molecules. The larger the flow rate and the higher the ionic strength, the faster the intercalated dyes are washed away from the DNAs. In the buffer with 1 M ionic strength, YOYO-1 and YO-PRO-1 are mostly washed away from DNA within 2 min of moderate buffer flow.
Topics: Fluorescent Dyes; DNA; Benzoxazoles; Osmolar Concentration
PubMed: 36917775
DOI: 10.1021/acs.jpcb.3c00777 -
European Biophysics Journal : EBJ Feb 2023In applications of bio-inspired nanoparticles (NPs), their composition is often optimised by including ionizable lipids. I use a generic statistical model to describe...
In applications of bio-inspired nanoparticles (NPs), their composition is often optimised by including ionizable lipids. I use a generic statistical model to describe the charge and potential distributions in lipid nanoparticles (LNPs) containing such lipids. The LNP structure is considered to contain the biophase regions separated by narrow interphase boundaries with water. Ionizable lipids are uniformly distributed at the biophase-water boundaries. The potential is there described at the mean-filed level combining the Langmuir-Stern equation for ionizable lipids and the Poisson-Boltzmann equation for other charges in water. The latter equation is used outside a LNP as well. With physiologically reasonable parameters, the model predicts the scale of the potential in a LNP to be rather low, smaller or about [Formula: see text], and to change primarily near the LNP-solution interface or, more precisely, inside an NP near this interface because the charge of ionizable lipids becomes rapidly neutralized along the coordinate towards the center of a LNP. The extent of dissociation-mediated neutralization of ionizable lipids along this coordinate increases but only slightly. Thus, the neutralization is primarily due to the negative and positive ions related to the ionic strength in solution and located inside a LNP.
Topics: Lipids; RNA, Small Interfering; Nanoparticles; Osmolar Concentration
PubMed: 36810604
DOI: 10.1007/s00249-023-01633-4 -
Scientific Reports Jun 2018The defensive slime of hagfish consists of a polyanionic mucin hydrogel that synergistically interacts with a fiber network forming a coherent and elastic hydrogel in...
The defensive slime of hagfish consists of a polyanionic mucin hydrogel that synergistically interacts with a fiber network forming a coherent and elastic hydrogel in high ionic strength seawater. In seawater, the slime deploys in less than a second entrapping large quantities of water by a well-timed thread skein unravelling and mucous gel swelling. This rapid and vast hydrogel formation is intriguing, as high ionic strength conditions generally counteract the swelling speed and ratio of polyelectrolyte hydrogels. In this work we investigate the effect of ionic strength and seawater cations on slime formation dynamics and functionality. In the absence of ionic strength skeins swell radially and unravel uncontrolled, probably causing tangling and creating a confined thread network that entraps limited water. At high ionic strength skeins unravel, but create a collapsed and dense fiber network. High ionic strength conditions therefore seem crucial for controlled skein unraveling, however not sufficient for water retention. Only the presence of naturally occurring Ca or Mg-ions allowed for an expanded network and full water retention probably due to Ca-mediated vesicle rupture and cross-linking of the mucin. Our study demonstrates that hagfish slime deployment is a well-timed, ionic-strength, and divalent-cation dependent dynamic hydrogel formation process.
Topics: Animals; Hagfishes; Mucins; Osmolar Concentration; Seawater
PubMed: 29959378
DOI: 10.1038/s41598-018-27975-0 -
Cellular Physiology and Biochemistry :... Feb 2021Volume regulated anion channels (VRACs) are ubiquitously expressed in all vertebrate cells. Despite many years of research, the fundamental mechanisms underlying VRAC... (Review)
Review
Volume regulated anion channels (VRACs) are ubiquitously expressed in all vertebrate cells. Despite many years of research, the fundamental mechanisms underlying VRAC activation are not understood. The recent molecular identification of the LRRC8 genes underlying VRAC revealed that VRACs are formed by a hexameric assembly of members of the LRRC8 gene family. Knowing the genes underlying VRACs allowed the discovery of novel VRAC functions into cell volume regulation, and first structure function studies revealed important insight in channel activation mechanisms. The determination of cryo-EM structures of homomeric LRRC8A and LRRC8D complexes provide a framework for a rational approach to investigate biophysical mechanisms. We discuss several recent advances within the structural framework, and we critically review the literature on the main mechanisms proposed to be involved in VRAC activation, including low intracellular ionic strength, membrane unfolding, oxidation, phosphorylation and G-protein coupling.
Topics: Animals; Cell Size; Humans; Membrane Proteins; Osmolar Concentration
PubMed: 33577730
DOI: 10.33594/000000329