17 research outputs found
Phase Separation Behavior of Supercharged Proteins and Polyelectrolytes
Membraneless organelles, like membrane-bound
organelles, are essential
to cell homeostasis and provide discrete cellular subcompartments.
Unlike classical organelles, membraneless organelles possess no physical
barrier but rather arise by phase separation of the organelle components
from the surrounding cytoplasm or nucleoplasm. Complex coacervation,
the liquid–liquid phase separation of oppositely charged polyelectrolytes,
is one of several phenomena that are hypothesized to drive the formation
and regulation of some membraneless organelles. Studies of the molecular
properties of globular proteins that drive complex coacervation are
limited as many proteins do not form complexes with oppositely charged
macromolecules at neutral pH and moderate ionic strengths. Protein
supercharging overcomes this problem and drives complexation with
oppositely charged macromolecules. In this work, several distinct
cationic supercharged green fluorescent protein (GFP) variants were
designed to examine the phase behavior with oppositely charged polyanionic
macromolecules. Cationic GFP variants phase separated with oppositely
charged macromolecules at various mixing ratios, salt concentrations,
and pH values. Efficient protein incorporation in the macromolecule
rich phase occurred over a range of protein and polymer mass fractions,
but the protein encapsulation efficiency was highest at the midpoint
of the phase separation regime. More positively charged proteins phase
separated over broader pH and salt ranges than those of proteins with
a lower charge density. Interestingly, each GFP variant phase separated
at higher salt concentrations with anionic synthetic macromolecules
compared to anionic biological macromolecules. Optical microscopy
revealed that most variants, depending on solution conditions, formed
liquid–liquid phase separations, except for GFP/DNA pairs that
formed solid aggregates under all tested conditions
Macro- and Microphase Separated Protein-Polyelectrolyte Complexes: Design Parameters and Current Progress
Protein-containing polyelectrolyte complexes (PECs) are a diverse class of materials, composed of two or more oppositely charged polyelectrolytes that condense and phase separate near overall charge neutrality. Such phase-separation can take on a variety of morphologies from macrophase separated liquid condensates, to solid precipitates, to monodispersed spherical micelles. In this review, we present an overview of recent advances in protein-containing PECs, with an overall goal of defining relevant design parameters for macro- and microphase separated PECs. For both classes of PECs, the influence of protein characteristics, such as surface charge and patchiness, co-polyelectrolyte characteristics, such as charge density and structure, and overall solution characteristics, such as salt concentration and pH, are considered. After overall design features are established, potential applications in food processing, biosensing, drug delivery, and protein purification are discussed and recent characterization techniques for protein-containing PECs are highlighted
Charge-Patterned Disordered Peptides Tune Intracellular Phase Separation in Bacteria
Subcellular
phase-separated compartments, known as biomolecular
condensates, play an important role in the spatiotemporal organization
of cells. To understand the sequence-determinants of phase separation
in bacteria, we engineered protein-based condensates in Escherichia coli using electrostatic interactions
as the main driving force. Minimal cationic disordered peptides were
used to supercharge negative, neutral, and positive globular model
proteins, enabling their phase separation with anionic biomacromolecules
in the cell. The phase behavior was governed by the interaction strength
between the cationic proteins and anionic biopolymers, in addition
to the protein concentration. The interaction strength primarily depended
on the overall net charge of the protein, but the distribution of
charge between the globular and disordered domains also had an impact.
Notably, the protein charge distribution between domains could tune
mesoscale attributes such as the size, number, and subcellular localization
of condensates within E. coli cells.
The length and charge density of the disordered peptides had significant
effects on protein expression levels, ultimately influencing the formation
of condensates. Taken together, charge-patterned disordered peptides
provide a platform for understanding the molecular grammar underlying
phase separation in bacteria
N‑Terminal Modification of Proteins with <i>o</i>‑Aminophenols
The
synthetic modification of proteins plays an important role
in chemical biology and biomaterials science. These fields provide
a constant need for chemical tools that can introduce new functionality
in specific locations on protein surfaces. In this work, an oxidative
strategy is demonstrated for the efficient modification of N-terminal
residues on peptides and N-terminal proline residues on proteins.
The strategy uses <i>o</i>-aminophenols or <i>o</i>-catechols that are oxidized to active coupling species <i>in
situ</i> using potassium ferricyanide. Peptide screening results
have
revealed that many N-terminal amino acids can participate in this
reaction, and that proline residues are particularly reactive. When
applied to protein substrates, the reaction shows a stronger requirement
for the proline group. Key advantages of the reaction include its
fast second-order kinetics and ability to achieve site-selective modification
in a single step using low concentrations of reagent. Although free
cysteines are also modified by the coupling reaction, they can be
protected through disulfide formation and then liberated after N-terminal
coupling is complete. This allows access to doubly functionalized
bioconjugates that can be difficult to access using other methods
Direct detection of nitrotyrosine-containing proteins using an aniline-based oxidative coupling strategy
A convenient two-step method is described for the detection of nitrotyrosine-containing proteins. First, nitrotyrosines are reduced to aminophenols using sodium dithionite. Following this, an oxidative coupling reaction is used to attach anilines bearing fluorescence reporters or affinity probes. Features of this approach include fast reaction times, pmol-level sensitivity, and excellent chemoselectivity
Multivalent Viral Capsids with Internal Cargo for Fibrin Imaging
<div><p>Thrombosis is the cause of many cardiovascular syndromes and is a significant contributor to life-threatening diseases, such as myocardial infarction and stroke. Thrombus targeted imaging agents have the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of thrombosis-related diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. In this work, we report the synthesis and use of chemically functionalized bacteriophage MS2 capsids as biomolecule-based nanoparticles for fibrin imaging. The capsids were modified using an oxidative coupling reaction, conjugating ∼90 copies of a fibrin targeting peptide to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents. The in vitro assessment of the capsids suggests that fibrin-targeted MS2 capsids could be used as delivery agents to thrombi for diagnostic or therapeutic applications.</p></div
Design of a viral capsid-based targeting system.
<p>(a) A scheme is shown for the synthesis of multivalent viral capsids for fibrin imaging. (b) Peptides used for attachment to MS2 or binding to fibrin. Peptides with a cysteine at the C-terminus were modified with a fluorescent dye and used for fibrin binding, while peptides with a C-terminal tyrosine were coupled to MS2 and tested for fibrin clotting inhibition. (c) Scheme for <i>o</i>-aminophenol peptide synthesis. The C-terminal tyrosine residue was coupled to 4-nitrobenzenediazonium tetrafluoroborate. The azo peptide was then cleaved from the resin and reduced to the <i>o</i>-aminophenol by sodium dithionite.</p
Characterization of MS2 conjugates.
<p>(a) The periodate-mediated oxidative coupling reaction takes place between <i>o</i>-aminophenol peptides and aniline containing MS2 capsids. (b) Alexa Fluor 680 and peptide-MS2 conjugates were analyzed by SDS-PAGE, with visualization of fluorescent (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100678#pone.0100678.s005" target="_blank">Figure S5</a>) and Coomassie-stained bands (shown). Lanes 1–3 show disassembled MS2 monomers labeled with Alexa Fluor 680. In lane 2, the GPR peptide was added and in lane 3 the GPS peptide was added. The upper bands represent the fraction of the MS2 monomers conjugated to the peptides. (c) Transmission electron microscopy, (d) dynamic light scattering, and (e) size-exclusion chromatography (fluorescence: λ<sub>ex</sub> = 280 nm, λ<sub>em</sub> = 330 nm) of MS2, GPR-MS2, and GPS-MS2 confirmed that the capsids remained intact after modification. Wide-field TEM images appear in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100678#pone.0100678.s007" target="_blank">Figure S7</a>.</p
Fibrin targeting with GPR-MS2 conjugates.
<p>(a) Five different agents were tested for fibrin binding properties. (b) Binding to fibrin monomers was confirmed with a clotting inhibition assay. Clotting times are plotted as the average of several trials (n = 4-6), shown with the standard error. The grey bar indicates the 95% confidence interval for the water control. At a concentration of 20 µM peptide, (222 nM MS2 capsid), only GPR-MS2 increased the thrombin induced clotting time of fibrin (Welch's t-test, p<0.05). (c) A representative near-infrared fluorescence image of fibrin clots in a 96-well plate indicated the <i>in vitro</i> binding of agents to the clots. (d) Binding was quantified, showing that the differences in relative spot intensity (normalized to the MS2 capsid intensity) between the agents were statistically significant (*p<0.05; **p<0.01). Normalized intensities are shown with the standard error.</p