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Scientific highlights 2015


Real-time observation of nonclassical protein crystallization kinetics


A. Sauter, F. Roosen-Runge, F. Zhang, G.Lotze, R.M.J. Jacobs and F.Schreiber,
SoftComp partner: Univ. Tuebingen
JACS 137 1485 (2015)


The crystallization of proteins is a major bottleneck of many projects in structural biology. While classical nucleation theory predicts the reversible aggregation of solute molecules and the formation nuclei with the density and structure of the crystals, features beyond the classical view have been proposed in the crystallization of proteins, colloids or clathrate hydrates.
A real-time study of protein crystallization using the model protein bovine β-lactoglobulin in the presence of CdCl2 has been performed using small-angle scattering (X-rays and neutrons) and optical microscopy.
The observation of the non-trivial crystallization kinetics can be explained by the following multistep mechanism: An intermediate structure within protein aggregates is formed first (indicated by a broad peak at ~0.7 nm-1 in the scattering data), followed by the formation of crystals within this intermediate phase. In this stage, the number of crystals increases with time while the crystal growth is rather slow within the dense intermediate phase due to the low mobility of proteins. In the next step, the intermediate phase is consumed by the nucleation of crystals and their growth. Finally, the crystals get exposed to the dilute phase. In this stage, the number of crystals stays almost constant, whereas the growth is more rapid due to the access to free protein molecules in the surrounding dilute phase.





Left: 3D plot of real-time SAXS data of a sample with 20 mg/ml BLG and 15 mM CdCl2. The intermediate structure shows in the broad peak at about 0.7 l/nm, Bragg peaks become visible later, whereas the broad peak disappears again.
Middle: Area under the broad peak ("intermediate"), area under the Bragg peaks ("Crystal") and I minus the other two contributions ("Liquid") as a function of time.
Right: SANS data of a sample with 33 mg/ml BLG and 17 mM CdCl2. Again, a broad peak becomes visible first, indicating the formation of a non-crystalline precursor-structure inside the protein aggregates. Subsequently, Bragg peaks become visible.


Glycans as Biofunctional Ligands for Gold Nanorods: Stability and Targeting in Protein-Rich Media


I. García, A. Sanchez-Iglesias, M. Henriksen-Lacey, M. Grzelczak, S. Penades and L. M. Liz-Marzań
SoftComp partner: CIC biomaGUNE
J. Amer. Chem. Soc. 137 (2015) 3686−3692
Article: http://pubs.acs.org/doi/abs/10.1021/jacs.5b01001


Poly(ethylene glycol) (PEG) has become the gold standard for stabilization of plasmonic nanoparticles (NPs) in biofluids, because it prevents aggregation while minimizing unspecific interactions with proteins. Application of Au NPs in biological environments requires the use of ligands that can target selected receptors, even in the presence of protein-rich media. We demonstrate that the stabilizing effect of low-molecular-weight glycans on both spherical and rod-like plasmonic NPs under physiological conditions, as bench-marked against the well-established PEG ligands. Glycan-coated NPs are resistant to adsorption of proteins from serum-containing media and avoid phagocytosis by macrophage-like cells, but retain selectivity toward carbohydrate-binding proteins in protein-rich biological media, such as tumoral cells. Given that mammalian lectins play an important role in a number of biological processes (innate immunity, leukocyte trafficking, modulation of cell-cell interactions, cell growth, etc.) our results support the idea that structures of complex and antigenic glycans onto plasmonic, anisotropic nanoparticles are suitable candidates for photothermal therapy on highly metastatic tumor cells, for instance, by blocking Gal-3 downstream biological processes.A real-time study of protein crystallization using the model protein bovine β-lactoglobulin in the presence of CdCl2 has been performed using small-angle scattering (X-rays and neutrons) and optical microscopy.
The observation of the non-trivial crystallization kinetics can be explained by the following multistep mechanism: An intermediate structure within protein aggregates is formed first (indicated by a broad peak at ~0.7 nm-1 in the scattering data), followed by the formation of crystals within this intermediate phase. In this stage, the number of crystals increases with time while the crystal growth is rather slow within the dense intermediate phase due to the low mobility of proteins. In the next step, the intermediate phase is consumed by the nucleation of crystals and their growth. Finally, the crystals get exposed to the dilute phase. In this stage, the number of crystals stays almost constant, whereas the growth is more rapid due to the access to free protein molecules in the surrounding dilute phase.





These results open the way toward the design of efficient therapeutic/diagnostic glycan-decorated plasmonic nanotools for specific biological applications.


The internal dynamics of fibrinogen and its implications for
coagulation and adsorption


S. Köhler, F. Schmid and G. Settanni
SoftComp partner: Univ. Mainz
PLOS Computational Biology
DOI:10.1371/journal.pcbi.1004346


Fibrinogen is a blood protein of vertebrates which plays an important role in the blood coagulation cascade. When activated, it aggregates and forms fibrin fibers, which are the basis of a blood clot. Clot persistence is regulated by plasmin, an enzyme which cuts fibrin fibers at specific places. A mechanistic understanding of fibrin degradation by plasmin is still missing. An important determinant of this process might be the flexibility of fibrinogen. For example, a great variety of conformations has been observed when fibrinogen adsorbs on material surfaces.

We have performed atomistic computer simulations that have helped to identify large bending motions occurring at specific hinges on fibrinogen. We could show how these bending motions can explain the variable conformations observed in experiments and how they help exposing sites where plasmin can cut fibrinogen. The bending and further cooperative effects observed in the simulations, thus, represent potential mechanisms for the regulation of blood clotting.





The figure represents several superimposed conformations of fibrinogen sampled along the simulations, highlighting the observed bending motions.


Directing Bacterial Motion by Structured Surfaces and Wall Slip


J. Hu, A. Wysocki, R.G. Winkler and G. Gompper
SoftComp partner: FZJ-Gompper
Sci. Rep. 5, 9586 (2015)
DOI: 10.1038/srep09586


The study and detailed understanding of the behavior of biological microswimmers, such as sperm, bacteria, and algae, is interesting for a better control and manipulaton of their motion, but also for the construction and optimization of artificial microswimmers for a variety of medical and technical tasks. Peritrichous bacteria, such as Escherichia coli and Salmonella, are propelled by a bundle of rotating helical flagella. Hydrodynamic interactions imply circular trajectories near surfaces.

We have constructed a detailed mechanical model of such a bacterium. Its motion in a fluid near a wall is studied by mesoscale hydrodynamics simulations. The curvature and orientation (clockwise, counterclockwise) of the trajectory depend on the fluid boundary conditions at the surface. A quantitative study reveals its quantitative dependence on the boundary slip length. These results are then employed to propose a novel approach to directing bacterial motion on striped surfaces with different slip lengths, which implies a transformation of the circular motion into a snaking motion along the stripe boundaries. The feasibility is demonstrated by a simulation of active Brownian rods, which also reveals a dependence of directional motion on the stripe width. This approach can be used for separating bacteria with different trajectory radii.




Swimming bacteria sense the fluid slip (characterized by the slip length b) of a nearby surface.


Ultrafast desorption of colloidal particles from fluid interfaces 



V. Poulichet and V. Garbin
SoftComp partner: Imperial College London
Proc. Natl. Acad. Sci. USA 112 (2015) 5932


Solid particles can replace surfactants to stabilize emulsions and foams. The attachment of particles onto drops and bubbles is typically considered to be irreversible because of a large energy barrier for particle detachment: millions of times the thermal energy for microparticles. As a result, significant challenges arise when destabilization and particle removal are a requirement. Here we demonstrate a method to promote the detachment of microparticles from bubbles using ultrasound. Ultrasound waves drive the bubbles into periodic compression-expansion, causing significant deformation and microstructural changes in the particle monolayer. Using high-speed microscopy we uncover different particle expulsion scenarios depending on the mode of bubble deformation, including highly directional patterns of particle release during shape oscillations. We identified conditions for complete particle removal and recovery in under a millisecond. Our method is programmable in time, and does not require any physicochemical modification of the fluids or the interface. This work addresses the emerging need for methods to recover interfacial particles from emulsions and foams in applications ranging from controlled release to interfacial catalysis and gas storage.




TOP: Ultrafast monolayer compression, buckling, and particle expulsion from a bubble stabilized by 3-µm particles undergoing compression-expansion in ultrasound at 50 kHz. BOTTOM: Patterned particle desorption for non-spherical bubble oscillations. Shape oscillations of a bubble coated with 500-nm particles, with a dominant four-fold mode developing over time and directing particle expulsion.


Selective flow-induced vesicle rupture to sort by membrane mechanical properties


A. Pommella, N. J. Brooks, J. M. Seddon, and V. Garbin
SoftComp partner: Imperial College London
Sci. Rep.5 (2015), 13163


Vesicle and cell rupture caused by large viscous stresses in ultrasonication is central to biomedical and bioprocessing applications. The flow-induced opening of lipid membranes can be exploited
to deliver drugs into cells, or to recover products from cells, provided that it can be obtained in a controlled fashion. Here we demonstrate that differences in lipid membrane and vesicle properties can enable selective flow-induced vesicle break-up. We obtained vesicle populations with different membrane properties by using different lipids (SOPC, DOPC, or POPC) and lipid:cholesterol mixtures (SOPC:chol and DOPC:chol). We subjected vesicles to large deformations in the acoustic microstreaming flow generated by ultrasound-driven microbubbles. By simultaneously deforming vesicles with different properties in the same flow, we determined the conditions in which rupture
is selective with respect to the membrane stretching elasticity. We also investigated the effect of vesicle radius and excess area on the threshold for rupture, and identified conditions for robust selectivity based solely on the mechanical properties of the membrane. Our work should enable new sorting mechanisms based on the difference in membrane composition and mechanical properties between different vesicles, capsules, or cells.



Vesicle break-up in the acoustic microstreaming flow generated by ultrasound-driven bubbles. Break-up is selective with respect to the membrane mechanical properties. Of two vesicles of identical size, only the DOPC vesicle breaks, while the DOPC:cholsesterol vesicle is intact in the same flow conditions. Selectivity is consistent with the difference in mechanical properties as shown in the graph.


Sensing Polymer Chain Dynamics through Ring Topology


S.Gooßen, M.Krutyeva, M.Sharp, A.Feoktystov, J.Allgaier, W.Pyckhout-Hintzen, A.Wischnewski and D.Richter
SoftComp partner: FZJ-Richter
Physical Review Letters
http://journals.aps.org/prl/accepted/04076Yb0Re91a157e4576dc089fc2a673c18ad237


By means of neutron spin echo spectroscopy, we show that the segmental dynamics of polymer rings immersed in linear chains is completely controlled by the host. Thus, rings are ideal probes for studying the entanglement dynamics of the embedding matrix. As a consequence of the unique ring topology, in long chain matrices the entanglement spacing is directly revealed, unaffected by local reptation of the host molecules beyond this distance. In shorter entangled matrices, where in the time frame of the experiment secondary effects such as contour length fluctuations (CLF) or constraint release (CR) could play a role, the ring motion reveals that CLF is weaker than assumed in state-of-the-art rheology and that CR is negligible. In conclusion, polymer rings have proved themselves to be a unique probe to investigate the matrix dynamics of long chains. We also anticipate that tube dilution effects prominent in branched architectures can, for example, be quantitatively addressed by a small number of immersed rings.



NSE data for a 20 kg/mol PEO ring in a deuterated 80 kg/mol linear matrix. Dashed black lines represent a fit to the data with the Rouse model for ring polymers. Solid colored lines indicate the plateaus related to a confinement size of 42 Å.
Last modified: 16/06/2015