19 research outputs found
Charging and Softening, Collapse, and Crystallization of Dipolar Phospholipid Membranes by Aqueous Ionic Liquid Solutions
Ionic
liquids have a variety of unique controllable structures
and properties. These properties may be used to tailor the self-assembly
of charged and dipolar biomolecules. Using solution X-ray scattering,
we measured the structure of DilaurylÂ(C<sub>12:0</sub>)-<i>sn</i>-glycero-3-phospho-l-choline (DLPC), a dipolar (or zwitterionic)
lipid, in the water-soluble room temperature ionic liquid Ethyl Methyl
Imidazolium Ethyl Sulfate (EMIES) and mixtures of EMIES and water.
We find that the interaction between the lipid bilayers is dominated
by the balance between the charging of the polar headgroups by the
ionic liquid, softening of the bilayer, and the osmotic pressure induced
by the solvent. This balance leads to the following changes with increasing
ionic liquid concentration: an incomplete unbinding transition from
an attractive regime to a swollen regime of the lamellar phase formed
by the bilayers. The swollen phase is followed by a collapse of the
bilayers into a highly desolvated lamellar phase at some critical
EMIES concentration, and eventually formation of lipid-crystalline
phase, at very high EMIES concentrations. The latter phase is revealed
by wide-angle X-ray scattering (WAXS) from the lipid solutions, showing
multiple Bragg peaks, consistent with highly ordered structures. These
structures were not observed in any other type of aqueous solutions
containing monovalent or multivalent ions. The kinetics and temperature
dependence of these transitions were also determined
Crystallization, Reentrant Melting, and Resolubilization of Virus Nanoparticles
Crystallization
is a fundamental and ubiquitous process that is
well understood in the case of atoms or small molecules, but its outcome
is still hard to predict in the case of nanoparticles or macromolecular
complexes. Controlling the organization of virus nanoparticles into
a variety of 3D supramolecular architectures is often done by multivalent
ions and is of great interest for biomedical applications such as
drug or gene delivery and biosensing, as well as for bionanomaterials
and catalysis. In this paper, we show that slow dialysis, over several
hours, of wild-type Simian Virus 40 (wt SV40) nanoparticle solution
against salt solutions containing MgCl<sub>2</sub>, with or without
added NaCl, results in wt SV40 nanoparticles arranged in a body cubic
center crystal structure with <i>Im</i>3<i>m</i> space group, as a thermodynamic product, in coexistence with soluble
wt SV40 nanoparticles. The nanoparticle crystals formed above a critical
MgCl<sub>2</sub> concentrations. Reentrant melting and resolubilization
of the virus nanoparticles took place when the MgCl<sub>2</sub> concentrations
passed a second threshold. Using synchrotron solution X-ray scattering
we determined the structures and the mass fraction of the soluble
and crystal phases as a function of MgCl<sub>2</sub> and NaCl concentrations.
A thermodynamic model, which balances the chemical potentials of the
Mg<sup>2+</sup> ions in each of the possible states, explains our
observations. The model reveals the mechanism of both the crystallization
and the reentrant melting and resolubilization and shows that counterion
entropy is the main driving force for both processes
Effect of Temperature on the Interactions between Dipolar Membranes
It is well-known that phospholipids in aqueous environment
self-assemble
into lamellar structures with a repeat distance governed by the interactions
between them. Yet, the understanding of these interactions is incomplete.
In this paper, we study the effect of temperature on the interlamellar
interactions between dipolar membranes. Using solution small-angle
X-ray scattering (SAXS), we measured the repeat distance between 1,2-dilauroyl-<i>sn</i>-glycero-3-phosphocholine (DLPC) bilayers at different
temperatures and osmotic stresses. We found that when no pressure
is applied the lamellar repeat distance, <i>D</i>, decreases
and then increases with increasing temperature. As the osmotic stress
increases, <i>D</i> decreases with temperature and then
increases to a limited extent, until at sufficiently high pressure <i>D</i> decreases with temperature in all the examined range.
We then reconstructed experimentally the equation of state and fit
it with a modified interaction model that takes into account the temperature
dependence of the fluctuation term. Finally, we showed how the thickness
of DLPC membranes decreases with temperature
Structure of Dynamic, Taxol-Stabilized, and GMPPCP-Stabilized Microtubule
Microtubule (MT)
is made of <i>αβ</i>-tubulin
heterodimers that dynamically assemble into a hollow nanotube composed
of straight protofilaments. MT dynamics is facilitated by hydrolysis
of guanosine-5′-triphosphate (GTP) and can be inhibited by
either anticancer agents like taxol or the nonhydrolyzable GTP analogues
like GMPPCP. Using high-resolution synchrotron X-ray scattering, we
have measured and analyzed the scattering curves from solutions of
dynamic MT (in other words, in the presence of excess GTP and free
of dynamic-inhibiting agents) and examined the effect of two MT stabilizers:
taxol and GMPPCP. Previously, we have analyzed the structure of dynamic
MT by docking the atomic model of tubulin dimer onto a 3-start left
handed helical lattice, derived from the PDB ID 3J6F. 3J6F corresponds to a
MT with 14 protofilaments. In this paper, we took into account the
possibility of having MT structures containing between 12 and 15 protofilaments.
MTs with 12 protofilaments were never observed. We determined the
radii, the pitch, and the distribution of protofilament number that
best fit the scattering data from dynamic MT or stabilized MT by taxol
or GMPPCP. We found that the protofilament number distribution shifted
when the MT was stabilized. Taxol increased the mass fraction of MT
with 13 protofilaments and decreased the mass fraction of MT with
14 protofilaments. GMPPCP reduced the mass fraction of MT with 15
protofilaments and increased the mass fraction of MT with 14 protofilaments.
The pitch, however, remained unchanged regardless of whether the MT
was dynamic or stabilized. Higher tubulin concentrations increased
the fraction of dynamic MT with 14 protofilaments
Reciprocal Grids: A Hierarchical Algorithm for Computing Solution X‑ray Scattering Curves from Supramolecular Complexes at High Resolution
In
many biochemical processes large biomolecular assemblies play
important roles. X-ray scattering is a label-free bulk method that can
probe the structure of large self-assembled complexes in solution.
As we demonstrate in this paper, solution X-ray scattering can measure
complex supramolecular assemblies at high sensitivity and resolution.
At high resolution, however, data analysis of larger complexes is
computationally demanding. We present an efficient method to compute
the scattering curves from complex structures over a wide range of
scattering angles. In our computational method, structures are defined
as hierarchical trees in which repeating subunits are docked into
their assembly symmetries, describing the manner subunits repeat in
the structure (in other words, the locations and orientations of the
repeating subunits). The amplitude of the assembly is calculated by
computing the amplitudes of the basic subunits on 3<i>D</i> reciprocal-space grids, moving up in the hierarchy, calculating
the grids of larger structures, and repeating this process for all
the leaves and nodes of the tree. For very large structures, we developed
a hybrid method that sums grids of smaller subunits in order to avoid
numerical artifacts. We developed protocols for obtaining high-resolution
solution X-ray scattering data from taxol-free microtubules at a wide
range of scattering angles. We then validated our method by adequately
modeling these high-resolution data. The higher speed and accuracy
of our method, over existing methods, is demonstrated for smaller
structures: short microtubule and tobacco mosaic virus. Our algorithm
may be integrated into various structure prediction computational
tools, simulations, and theoretical models, and provide means for
testing their predicted structural model, by calculating the expected
X-ray scattering curve and comparing with experimental data
RNA Encapsidation by SV40-Derived Nanoparticles Follows a Rapid Two-State Mechanism
Remarkably, uniform virus-like particles self-assemble
in a process
that appears to follow a rapid kinetic mechanism. The mechanisms by
which spherical viruses assemble from hundreds of capsid proteins
around nucleic acid, however, are yet unresolved. Using time-resolved
small-angle X-ray scattering (TR-SAXS), we have been able to directly
visualize SV40 VP1 pentamers encapsidating short RNA molecules (500mers).
This assembly process yields <i>T</i> = 1 icosahedral particles comprised of 12 pentamers and one RNA molecule.
The reaction is nearly one-third complete within 35 ms, following
a two-state kinetic process with no detectable intermediates. Theoretical
analysis of kinetics, using a master equation, shows that the assembly
process nucleates at the RNA and continues by a cascade of elongation
reactions in which one VP1 pentamer is added at a time, with a rate
of approximately 10<sup>9</sup> M<sup>–1</sup> s<sup>–1</sup>. The reaction is highly robust and faster than the predicted diffusion
limit. The emerging molecular mechanism, which appears to be general
to viruses that assemble around nucleic acids, implicates long-ranged
electrostatic interactions. The model proposes that the growing nucleo-protein
complex acts as an electrostatic antenna that attracts other capsid
subunits for the encapsidation process
Nano-Drugs Based on Nano Sterically Stabilized Liposomes for the Treatment of Inflammatory Neurodegenerative Diseases
<div><p>The present study shows the advantages of liposome-based nano-drugs as a novel strategy of delivering active pharmaceutical ingredients for treatment of neurodegenerative diseases that involve neuroinflammation. We used the most common animal model for multiple sclerosis (MS), mice experimental autoimmune encephalomyelitis (EAE). The main challenges to overcome are the drugs’ unfavorable pharmacokinetics and biodistribution, which result in inadequate therapeutic efficacy and in drug toxicity (due to high and repeated dosage). We designed two different liposomal nano-drugs, i.e., nano sterically stabilized liposomes (NSSL), remote loaded with: (a) a “water-soluble” amphipathic weak acid glucocorticosteroid prodrug, methylprednisolone hemisuccinate (MPS) or (b) the amphipathic weak base nitroxide, Tempamine (TMN). For the NSSL-MPS we also compared the effect of passive targeting alone and of active targeting based on short peptide fragments of ApoE or of β-amyloid. Our results clearly show that for NSSL-MPS, active targeting is not superior to passive targeting. For the NSSL-MPS and the NSSL-TMN it was demonstrated that these nano-drugs ameliorate the clinical signs and the pathology of EAE. We have further investigated the MPS nano-drug’s therapeutic efficacy and its mechanism of action in both the acute and the adoptive transfer EAE models, as well as optimizing the perfomance of the TMN nano-drug. The highly efficacious anti-inflammatory therapeutic feature of these two nano-drugs meets the criteria of disease-modifying drugs and supports further development and evaluation of these nano-drugs as potential therapeutic agents for diseases with an inflammatory component.</p></div
Comparison of passively targeted NSSL and actively targeted peptide-conjugated NSSL.
<p><b>(A)</b> Representative fluorescent microscopy images comparing brain accumulation of NSSL and their payload as is (A, A1), β-amyloid NSSL(B,B1), and ApoE NSSL (C,C1) in healthy mice brain showing an increase in the amount of actively targeted NSSL and their payload accumulating, compared to passively targeted NSSL. <b>(B)</b> Comparison of the therapeutic efficacy of passively targeted NSSL-MPS and actively targeted peptide-conjugated NSSL-MPS in the acute EAE mice model. SJL mice were treated by IV injections on days 10, 12, 14 post-immunization with saline (control) (◆), NSSL-MPS (●), Apo-E NSSL-MPS (▲) or β-amyloid NSSL-MPS (<b>■</b>). * p-value < 0.0001.</p
Comparison of the therapeutic efficacy of NSSL-MPS and free MPS in the adoptive transfer EAE mice model.
<p><sup>a</sup> Significant difference from the control group P<0.000001</p><p><sup>b</sup> Significant difference from the NSSL-MPS group P<0.0005</p><p><sup>c</sup> Significant difference from the control group P<0.00005</p><p><sup>d</sup> Significant difference from the NSSL-MPS group P<0.005.</p><p>Comparison of the therapeutic efficacy of NSSL-MPS and free MPS in the adoptive transfer EAE mice model.</p