6 research outputs found
Renewal of the Air–Water Interface as a Critical System Parameter of Protein Stability: Aggregation of the Human Growth Hormone and Its Prevention by Surface-Active Compounds
Soluble proteins are often highly
unstable under mixing conditions
that involve dynamic contacting between the main liquid phase and
a gas phase. The recombinant human growth hormone (rhGH) was recently
shown to undergo aggregation into micrometer-sized solid particles
composed of non-native (mis- or unfolded) protein, once its solutions
were stirred or shaken to generate a continuously renewed air–water
interface. To gain deepened understanding and improved quantification
of the air–water interface effect on rhGH stability, we analyzed
the protein’s aggregation rate (<i>r</i><sub>agg</sub>) at controlled specific air–water surface areas (<i>a</i><sub>G/L</sub>) established by stirring or bubble aeration.
We show that in spite of comparable time-averaged values for <i>a</i><sub>G/L</sub> (≈ 100 m<sup>2</sup>/m<sup>3</sup>), aeration gave a 40-fold higher <i>r</i><sub>agg</sub> than stirring. The enhanced <i>r</i><sub>agg</sub> under
aeration was ascribed to faster macroscopic regeneration of free <i>a</i><sub>G/L</sub> during aeration as compared to stirring.
We also show that <i>r</i><sub>agg</sub> was independent
of the rhGH concentration in the range 0.67 – 6.7 mg/mL, and
that it increased linearly dependent on the available <i>a</i><sub>G/L</sub>. The nonionic surfactant Pluronic F-68, added in 1.6-fold
molar excess over rhGH present, resulted in complete suppression of <i>r</i><sub>agg</sub>. Foam formation was not a factor influencing <i>r</i><sub>agg</sub>. Using analysis by circular dichroism spectroscopy
and small-angle X-ray scattering, we show that in the presence of
Pluronic F-68 under both stirring and aeration, the soluble protein
retained its original fold, featuring native-like relative composition
of secondary structural elements. We further provide evidence that
the efficacy of Pluronic F-68 resulted from direct, probably hydrophobic
protein–surfactant interactions that prevented rhGH from becoming
attached to the air–water interface. Surface-induced aggregation
of rhGH is suggested to involve desorption of non-native protein from
the air–water interface as the key limiting step. Proteins
or protein aggregates released back into the bulk liquid appear to
be essentially insoluble
Softness of Atherogenic Lipoproteins: A Comparison of Very Low Density Lipoprotein (VLDL) and Low Density Lipoprotein (LDL) Using Elastic Incoherent Neutron Scattering (EINS)
Apolipoprotein B100 (apoB100)-containing plasma lipoproteins (LDL and VLDL) supply tissues and cells with cholesterol and fat. During lipolytic conversion from VLDL to LDL the size and chemical composition of the particles change, but the apoB100 molecule remains bound to the lipids and regulates the receptor mediated uptake. The molecular physical parameters which control lipoprotein remodeling and enable particle stabilization by apoB100 are largely unknown. Here, we have compared the molecular dynamics and elasticities of VLDL and LDL derived by elastic neutron scattering temperature scans. We have determined thermal motions, dynamical transitions, and molecular fluctuations, which reflect the temperature-dependent motional coupling between lipid and protein. Our results revealed that lipoprotein particles are extremely soft and flexible. We found substantial differences in the molecular resiliences of lipoproteins, especially at higher temperatures. These discrepancies not only can be explained in terms of lipid composition and mobility but also suggest that apoB100 displays different dynamics dependent on the lipoprotein it is bound to. Hence, we suppose that the inherent conformational flexibility of apoB100 permits particle stabilization upon lipid exchange, whereas the dynamic coupling between protein and lipids might be a key determinant for lipoprotein conversion and atherogenicity
Peptides at the Interface: Self-Assembly of Amphiphilic Designer Peptides and Their Membrane Interaction Propensity
Self-assembling
amphiphilic designer peptides have been successfully
applied as nanomaterials in biomedical applications. Understanding
molecular interactions at the peptide–membrane interface is
crucial, since interactions at this site often determine (in)Âcompatibility.
The present study aims to elucidate how model membrane systems of
different complexity (in particular single-component phospholipid
bilayers and lipoproteins) respond to the presence of amphiphilic
designer peptides. We focused on two short anionic peptides, V<sub>4</sub>WD<sub>2</sub> and A<sub>6</sub>YD, which are structurally
similar but showed a different self-assembly behavior. A<sub>6</sub>YD self-assembled into high aspect ratio nanofibers at low peptide
concentrations, as evidenced by synchrotron small-angle X-ray scattering
and electron microscopy. These supramolecular assemblies coexisted
with membranes without remarkable interference. In contrast, V<sub>4</sub>WD<sub>2</sub> formed only loosely associated assemblies over
a large concentration regime, and the peptide promoted concentration-dependent
disorder on the membrane arrangement. Perturbation effects were observed
on both membrane systems although most likely induced by different
modes of action. These results suggest that membrane activity critically
depends on the peptide’s inherent ability to form highly cohesive
supramolecular structures
Sketch of the experimental setup.
<p>The experimental arrangement for time-resolved X-ray measurements at the Austrian SAXS–beamline at the ELETTRA synchrotron light source is shown. For T-jump experiments, an erbium laser beam (IR), wavelength λ = 1.5 µm, was directed via a prism onto the sample capillary which was thermostated with a Peltier unit. Laser pulse energy was 2 J within 2 ms resulting in an average T-jump amplitude of 10–12°C. The exposure time was 10 ms per frame. For T-drop experiments, the empty X-ray capillary was pre-cooled in a stream of nitrogen adjusted to −20°C. LDL samples, preheated to approx.10°C above the melting transition, were injected by a motor-driven syringe. A drop in temperature of about 20°C could be induced in about 3–4 s. The exposure time was 250 ms per frame.</p
Time-resolved nanophase transition in LDL.
<p>The rise in the integrated intensity of the 1st side-maximum upon laser jump is shown as a function of time (A). The time slicing was 10 ms per image. The time point of laser flash is set to zero seconds. The error function of statistical variation displays a maximum inaccuracy in time of about 5 ms. Thus, the offset in transition is much shorter than the sampling time of 10 ms and the 2 ms of laser flash. The integrated intensities of the 1st side-maximum obtained by static measurements within a temperature range of 0°C and 50°C with a step width of 5°C (B, left panel) are correlated to the time-course of integrated intensities of the 1st side-maximum obtained by dynamic measurements (B, right panel). For static measurements, a measuring time of 30 s and an equilibration time of 10 minutes at each temperature was chosen. For dynamic measurements, the measuring time per frame was 250 ms. A half-time of 2 seconds, corresponding to a temperature drop of about 10°C, could be achieved to pass through the transition temperature. The decline in integrated intensity strictly followed the drop in temperature. Tm for the LDL sample shown was about 22°C, as determined by microcalorimetry.</p
Interleukin-10: An Anti-Inflammatory Marker To Target Atherosclerotic Lesions via PEGylated Liposomes
Atherosclerosis (AS) causes cardiovascular disease, which
leads
to fatal clinical end points like myocardial infarction or stroke,
the most prevalent causes of death in developed countries. An early,
noninvasive method of detection and diagnosis of atherosclerotic lesions
is necessary to prevent and treat these clinical end points. Working
toward this goal, we examined recombinant interleukin-10 (IL-10),
stealth liposomes with nanocargo potency for NMRI relevant contrast
agents, and IL-10 coupled to stealth liposomes in an ApoE-deficient
mouse model using confocal laser-scanning microscopy (CLSM). Through <i>ex vivo</i> incubation and imaging with CLSM, we showed that
fluorescently labeled IL-10 is internalized by AS plaques, and a low
signal is detected in both the less injured aortic surfaces and the
arteries of wild-type mice. <i>In vivo</i> experiments included
intravenous injections of (i) fluorescent IL-10, (ii) IL-10 targeted
carboxyfluorescin (CF−) labeled stealth liposomes, and (iii)
untargeted CF-labeled stealth liposomes. Twenty-four hours after injection
the arteries were dissected and imaged <i>ex vivo</i>. Compared
to free IL-10, we observed a markedly stronger fluorescence intensity
with IL-10 targeted liposomes at AS plaque regions. Moreover, untargeted
CF-labeled liposomes showed only weak, unspecific binding. Neither
free IL-10 nor IL-10 targeted liposomes showed significant immune
reaction when injected into wild-type mice. Thus, the combined use
of specific anti-inflammatory proteins, high payloads of contrast
agents, and liposome particles should enable current imaging techniques
to better recognize and visualize AS plaques for research and prospective
therapeutic strategies