scholarly journals Intracellular Ionic Strength Sensing Using NanoLuc

2021 ◽  
Vol 22 (2) ◽  
pp. 677
Author(s):  
Tausif Altamash ◽  
Wesam Ahmed ◽  
Saad Rasool ◽  
Kabir H. Biswas
Keyword(s):  
Thermal Stability ◽  
Phase Separation ◽  
Drug Discovery ◽  
Ionic Strength ◽  
Cellular Signaling ◽  
Live Cells ◽  
Protein Activity ◽  
Cellular Survival ◽  
Cellular Processes ◽  
Wide Range

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.

scholarly journals Current Understanding of Molecular Phase Separation in Chromosomes

2021 ◽  
Vol 22 (19) ◽  
pp. 10736
Author(s):  
Je-Kyung Ryu ◽  
Da-Eun Hwang ◽  
Jeong-Mo Choi
Keyword(s):  
Phase Separation ◽  
Computational Methods ◽  
Current Understanding ◽  
Study Phase ◽  
Cellular Processes ◽  
Wide Range ◽  
Recent Phase ◽  
Separation Model

Biomolecular phase separation denotes the demixing of a specific set of intracellular components without membrane encapsulation. Recent studies have found that biomolecular phase separation is involved in a wide range of cellular processes. In particular, phase separation is involved in the formation and regulation of chromosome structures at various levels. Here, we review the current understanding of biomolecular phase separation related to chromosomes. First, we discuss the fundamental principles of phase separation and introduce several examples of nuclear/chromosomal biomolecular assemblies formed by phase separation. We also briefly explain the experimental and computational methods used to study phase separation in chromosomes. Finally, we discuss a recent phase separation model, termed bridging-induced phase separation (BIPS), which can explain the formation of local chromosome structures.

scholarly journals Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Daesun Song ◽  
Yongsang Jo ◽  
Jeong-Mo Choi ◽  
Yongwon Jung
Keyword(s):  
Phase Separation ◽  
Protein Interactions ◽  
Cellular Membrane ◽  
Formation Mechanisms ◽  
Cellular Processes ◽  
Quantitative Measurements ◽  
Wide Range ◽  
Client Proteins ◽  
Spatiotemporal Control

Abstract Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liquid–liquid phase separation, biomolecular behaviors such as protein interactions inside these liquid compartments have been largely unexplored. Here we report quantitative measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase separation conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase separation model, we discovered that the operational proximity of clients (measured from client–client interactions) could be over 16 times higher than the expected proximity from actual client concentrations inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase separation compartmentalization: (1) clients are selectively recruited into compartments, leading to concentration enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.

Calculating Melting Temperature of Native and Modified Oligonucleotide Complexes at Various Cation Concentrations with the Use of Enhanced Counterion Condensation Model

2010 ◽  
Vol 3 (2) ◽  
pp. 61-75
Author(s):  
Aleksandr A. Lomzov ◽  
Dmitriy V. Pyshnyi
Keyword(s):  
Thermal Stability ◽  
Ionic Strength ◽  
Melting Temperature ◽  
Binding Constants ◽  
Data Sets ◽  
Dna Duplexes ◽  
Counterion Binding ◽  
Cation Concentration ◽  
Wide Range ◽  
Thermal Stability Of

A new model describing the influence of ionic strength on thermal stability of DNA comlexes of oligonucleotides is proposed. This model assumes that binding of cations with DNA polyanions influences solely the entropy of hybridization and has a saturating mode. The efficacy of counterion binding with single- and double-stranded DNA is different, and the number of cations which bind additionally with the oligonucleotide at duplex formation depends on bulk cation concentration. Analytical equations describing the influence of cation concentration on melting temperature of DNA-duplexes as function of the length of oligonucleotide, its GC-composition and presence of the modification (non-nucleotide insert) were obtained. The values of melting temperature (Tm ) and thermodynamic parameters ( o ∆H , o ∆S ) characterizing the hybridization of both native and «bridged» oligonucleotides (bearing non-nucleotide insert on the basis of diethylene glycol phosphodiester) with DNA in various concentrations of NaCl (0,01÷1 М) were obtained using the UV-melting technique. Based on both the data obtained and presented in literature the database (695 data sets) characterizing the influence of ionic strength on the thermal stability of oligonucleotide complexes of various structure is developed. The database analysis allows us to obtain the values of the equilibrium binding constants for condensation of caions on DNA and the number of ions required for saturation of a discrete binding site. The proposed enhanced model of cation condensation utilizing unified thermodynamic increments of dsDNA formation allows us to calculate melting temperatures of DNAduplexes in the wide range of ionic strength ([Na+] = 0,01÷1 М) with high accuracy.

scholarly journals SARS-CoV-2 infection remodels the host protein thermal stability landscape

2020 ◽  
Author(s):  
Joel Selkrig ◽  
Megan Stanifer ◽  
André Mateus ◽  
Karin Mitosch ◽  
Inigo Barrio-Hernandez ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Rna Splicing ◽  
Host Protein ◽  
Effective Vaccine ◽  
Global Changes ◽  
Human Pathogens ◽  
Protein Activity ◽  
Cellular Processes ◽  
Wide Scale ◽  
Global Threat

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS-CoV-2 hijacks host cellular machineries on a systems-wide scale so that potential host-directed therapy can be developed. In situ proteome-wide abundance and thermal stability measurements using thermal proteome profiling (TPP), can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS-CoV-2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and regulation of RNA splicing. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS-CoV-2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS-CoV-2 infection.

scholarly journals Tunable thermo-reversible bicontinuous nanoparticle gel driven by the binary solvent segregation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuyin Xi ◽  
Ronald S. Lankone ◽  
Li-Piin Sung ◽  
Yun Liu
Keyword(s):  
Phase Separation ◽  
Domain Size ◽  
Binary Solvent ◽  
Colloidal Systems ◽  
Colloidal Assembly ◽  
Structures And Properties ◽  
Wide Range ◽  
Equilibrium Process ◽  
Equilibrium Structures ◽  
Non Equilibrium

AbstractBicontinuous porous structures through colloidal assembly realized by non-equilibrium process is crucial to various applications, including water treatment, catalysis and energy storage. However, as non-equilibrium structures are process-dependent, it is very challenging to simultaneously achieve reversibility, reproducibility, scalability, and tunability over material structures and properties. Here, a novel solvent segregation driven gel (SeedGel) is proposed and demonstrated to arrest bicontinuous structures with excellent thermal structural reversibility and reproducibility, tunable domain size, adjustable gel transition temperature, and amazing optical properties. It is achieved by trapping nanoparticles into one of the solvent domains upon the phase separation of the binary solvent. Due to the universality of the solvent driven particle phase separation, SeedGel is thus potentially a generic method for a wide range of colloidal systems.

scholarly journals Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yi Zhang ◽  
Kyle Brown ◽  
Yucong Yu ◽  
Ziad Ibrahim ◽  
Mohamad Zandian ◽  
...  
Keyword(s):  
Phase Separation ◽  
Cell Nucleus ◽  
Active Site ◽  
Histone Acetyltransferase ◽  
Histone H3 ◽  
Catalytic Core ◽  
Cellular Processes ◽  
Storage Pool ◽  
Core Components ◽  
Acetyltransferase P300

AbstractThe transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.

Synthesis of sp3-Enriched β-Fluoro Sulfonyl Chlorides

Synthesis ◽  
2020 ◽  
Author(s):  
Oleksandr O. Grygorenko ◽  
Rustam Gurbanov ◽  
Andriy Sokolov ◽  
Sergey Golovach ◽  
Kostiantyn Melnykov ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Double Bond ◽  
Building Blocks ◽  
Protecting Groups ◽  
Sulfonyl Chlorides ◽  
Wide Range ◽  
Cyclic Compounds

AbstractA three-step approach to the synthesis of sp3-enriched β-fluoro sulfonyl chlorides starting from alkenes is reported. The method was successfully applied to a wide range of acyclic and cyclic substrates, bearing either an exocyclic or an endocyclic double bond. The procedure worked with a wide range of substrates and tolerated a number of functional and protecting groups. Moreover, the target cyclic compounds were obtained as single cis diastereomers on a multigram scale. The title compounds are promising building blocks for drug discovery that can be used to obtain sp3-enriched β-fluoro and α,β-unsaturated sulfonamides.

scholarly journals Emerging Silk Material Trends: Repurposing, Phase Separation and Solution-Based Designs

Materials ◽  
2021 ◽  
Vol 14 (5) ◽  
pp. 1160
Author(s):  
F. Philipp Seib
Keyword(s):  
Phase Separation ◽  
Recent Progress ◽  
Fiber Formation ◽  
Live Cells ◽  
Specific Reference ◽  
New Developments ◽  
Silk Fabrics ◽  
Induced Crystallization ◽  
Liquid Liquid Phase Separation

Silk continues to amaze. This review unravels the most recent progress in silk science, spanning from fundamental insights to medical silks. Key advances in silk flow are examined, with specific reference to the role of metal ions in switching silk from a storage to a spinning state. Orthogonal thermoplastic silk molding is described, as is the transfer of silk flow principles for the triggering of flow-induced crystallization in other non-silk polymers. Other exciting new developments include silk-inspired liquid–liquid phase separation for non-canonical fiber formation and the creation of “silk organelles” in live cells. This review closes by examining the role of silk fabrics in fashioning facemasks in response to the SARS-CoV-2 pandemic.

scholarly journals Glossary and tutorial of xenobiotic metabolism terms used during small molecule drug discovery and development (IUPAC Technical Report)

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Paul Erhardt ◽  
Kenneth Bachmann ◽  
Donald Birkett ◽  
Michael Boberg ◽  
Nicholas Bodor ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Small Molecule ◽  
Chemical Structure ◽  
Early Stage ◽  
Xenobiotic Metabolism ◽  
Drug Discovery And Development ◽  
Small Molecule Drug ◽  
Technical Report ◽  
Wide Range ◽  
Draft Version

Abstract This project originated more than 15 years ago with the intent to produce a glossary of drug metabolism terms having definitions especially applicable for use by practicing medicinal chemists. A first-draft version underwent extensive beta-testing that, fortuitously, engaged international audiences in a wide range of disciplines involved in drug discovery and development. It became clear that the inclusion of information to enhance discussions among this mix of participants would be even more valuable. The present version retains a chemical structure theme while expanding tutorial comments that aim to bridge the various perspectives that may arise during interdisciplinary communications about a given term. This glossary is intended to be educational for early stage researchers, as well as useful for investigators at various levels who participate on today’s highly multidisciplinary, collaborative small molecule drug discovery teams.

Evaluation of effects of pH and ionic strength on colloidal stability of IgG solutions by PEG-induced liquid-liquid phase separation

2016 ◽  
Vol 145 (18) ◽  
pp. 185101 ◽  
Author(s):  
Ronald W. Thompson ◽  
Ramil F. Latypov ◽  
Ying Wang ◽  
Aleksey Lomakin ◽  
Julie A. Meyer ◽  
...  
Keyword(s):  
Phase Separation ◽  
Ionic Strength ◽  
Liquid Phase ◽  
Colloidal Stability ◽  
Liquid Phase Separation ◽  
Effects Of Ph ◽  
Liquid Liquid Phase Separation
Sign in / Sign up

Export Citation Format

Share Document