scholarly journals Ionic polypeptide tags for protein phase separation

2019 ◽  
Vol 10 (9) ◽  
pp. 2700-2707 ◽  
Author(s):  
Rachel A. Kapelner ◽  
Allie C. Obermeyer
Keyword(s):  
Phase Separation ◽  
Globular Proteins ◽  
Complex Coacervation ◽  
Protein Activity ◽  
Physiological Conditions

Short ionic polypeptide tags were demonstrated to drive complex coacervation of globular proteins at physiological conditions while maintaining protein activity.

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.

Aggregation, gelation and phase separation of heat denatured globular proteins

2002 ◽  
Vol 304 (1-2) ◽  
pp. 253-265 ◽  
Author(s):  
Dominique Durand ◽  
Jean Christophe Gimel ◽  
Taco Nicolai
Keyword(s):  
Phase Separation ◽  
Globular Proteins

scholarly journals Prebiotically-relevant low polyion multivalency can improve functionality of membraneless compartments

2020 ◽  
Author(s):  
Fatma Pir Cakmak ◽  
Saehyun Choi ◽  
McCauley O. Meyer ◽  
Philip C. Bevilacqua ◽  
Christine D. Keating
Keyword(s):  
Molecular Weight ◽  
Phase Separation ◽  
Structure Formation ◽  
Rna Structure ◽  
Salt Resistance ◽  
Origins Of Life ◽  
Complex Coacervation ◽  
The Impact ◽  
Local Ph

AbstractMultivalent polyions can undergo complex coacervation, producing membraneless compartments that accumulate ribozymes and enhance catalysis, and offering a mechanism for functional prebiotic compartmentalization in the origins of life. Here, we evaluated the impact of low, prebiotically-relevant polyion multivalency in coacervate performance as functional compartments. As model polyions, we used positively and negatively charged homopeptides with one to 100 residues, and adenosine mono-, di-, and triphosphate nucleotides. Polycation/polyanion pairs were tested for coacervation, and resulting membraneless compartments were analyzed for salt resistance, ability to provide a distinct internal microenvironment (apparent local pH, RNA partitioning), and effect on RNA structure formation. We find that coacervates formed by phase separation of the relatively shorter polyions more effectively generated distinct pH microenvironments, accumulated RNA, and preserved duplexes. Hence, reduced multivalency polyions are not only viable as functional compartments for prebiotic chemistries, but they can offer advantages over higher molecular weight analogues.

scholarly journals TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau

2021 ◽  
Vol 118 (9) ◽  
pp. e2014188118
Author(s):  
Peter E. A. Ash ◽  
Shuwen Lei ◽  
Jenifer Shattuck ◽  
Samantha Boudeau ◽  
Yari Carlomagno ◽  
...  
Keyword(s):  
Phase Separation ◽  
Polyethylene Glycol ◽  
Stress Response ◽  
Tau Protein ◽  
Rna Binding ◽  
Biological Activities ◽  
Rna Binding Protein ◽  
Physiological Conditions ◽  
Tau Oligomers

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

scholarly journals Sequence Grammar Underlying Unfolding and Phase Separation of Globular Proteins

2021 ◽  
Author(s):  
Kiersten M. Ruff ◽  
Yoon Hee Choi ◽  
Dezerae Cox ◽  
Angelique R. Ormsby ◽  
Yoochan Myung ◽  
...  
Keyword(s):  
Phase Separation ◽  
Globular Proteins

scholarly journals Prebiotically-relevant low polyion multivalency can improve functionality of membraneless compartments

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Fatma Pir Cakmak ◽  
Saehyun Choi ◽  
McCauley O. Meyer ◽  
Philip C. Bevilacqua ◽  
Christine D. Keating
Keyword(s):  
Molecular Weight ◽  
Phase Separation ◽  
Structure Formation ◽  
Rna Structure ◽  
Functional Performance ◽  
Salt Resistance ◽  
Origins Of Life ◽  
Complex Coacervation ◽  
The Impact ◽  
Local Ph

AbstractMultivalent polyions can undergo complex coacervation, producing membraneless compartments that accumulate ribozymes and enhance catalysis, and offering a mechanism for functional prebiotic compartmentalization in the origins of life. Here, we evaluate the impact of lower, more prebiotically-relevant, polyion multivalency on the functional performance of coacervates as compartments. Positively and negatively charged homopeptides with 1–100 residues and adenosine mono-, di-, and triphosphate nucleotides are used as model polyions. Polycation/polyanion pairs are tested for coacervation, and resulting membraneless compartments are analyzed for salt resistance, ability to provide a distinct internal microenvironment (apparent local pH, RNA partitioning), and effect on RNA structure formation. We find that coacervates formed by phase separation of the shorter polyions more effectively generated distinct pH microenvironments, accumulated RNA, and preserved duplexes than those formed by longer polyions. Hence, coacervates formed by reduced multivalency polyions are not only viable as functional compartments for prebiotic chemistries, they can outperform higher molecular weight analogues.

scholarly journals Phase separation and dynamical arrest for particles interacting with mixed potentials—the case of globular proteins revisited

Soft Matter ◽  
2011 ◽  
Vol 7 (3) ◽  
pp. 857-860 ◽  
Author(s):  
Thomas Gibaud ◽  
Frédéric Cardinaux ◽  
Johan Bergenholtz ◽  
Anna Stradner ◽  
Peter Schurtenberger
Keyword(s):  
Phase Separation ◽  
Globular Proteins ◽  
Mixed Potentials

scholarly journals Critical adsorption of multiple polyelectrolytes onto a nanosphere: splitting the adsorption–desorption transition boundary

2020 ◽  
Vol 17 (167) ◽  
pp. 20200199 ◽  
Author(s):  
Daniel L. Z. Caetano ◽  
Sidney J. de Carvalho ◽  
Ralf Metzler ◽  
Andrey G. Cherstvy
Keyword(s):  
Phase Separation ◽  
Surface Charge Density ◽  
Globular Proteins ◽  
Spherical Nanoparticles ◽  
Separation Curve ◽  
Low Salt ◽  
Electrostatic Binding ◽  
Adsorption Desorption ◽  
Oppositely Charged ◽  
Added Salt

Employing extensive Monte Carlo computer simulations, we investigate in detail the properties of multichain adsorption of charged flexible polyelectrolytes (PEs) onto oppositely charged spherical nanoparticles (SNPs). We quantify the conditions of critical adsorption—the phase-separation curve between the adsorbed and desorbed states of the PEs—as a function of the SNP surface-charge density and the concentration of added salt. We study the degree of fluctuations of the PE–SNP electrostatic binding energy, which we use to quantify the emergence of the phase subtransitions, including a series of partially adsorbed PE configurations. We demonstrate how the phase-separation adsorption–desorption boundary shifts and splits into multiple subtransitions at low-salt conditions, thereby generalizing and extending the results for critical adsorption of a single PE onto the SNP. The current findings are relevant for finite concentrations of PEs around the attracting SNP, such as the conditions for PE adsorption onto globular proteins carrying opposite electric charges.

scholarly journals Biodegradable zwitterionic nanoparticles with tunable UCST-type phase separation under physiological conditions

Nanoscale ◽  
2019 ◽  
Vol 11 (35) ◽  
pp. 16582-16591 ◽  
Author(s):  
Mattia Sponchioni ◽  
Paola Rodrigues Bassam ◽  
Davide Moscatelli ◽  
Paolo Arosio ◽  
Umberto Capasso Palmiero
Keyword(s):  
Phase Separation ◽  
Controlled Delivery ◽  
Physiological Conditions ◽  
Type Phase ◽  
Delivery Strategies

We report tunable biodegradable zwitterionic nanoparticles with UCST behavior under physiological conditions that can be used in controlled delivery strategies.

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