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Scientific highlights 2016


Dramatic influence of patchy attractions on short-time protein diffusion under crowded conditions


S.Bucciarelli, J.S.Myung, B.Farago, S.Das, G.Vliegenthart, O.Holderer, R.G.Winkler, P.Schurtenberger, G.Gompper and A.Stradner
SoftComp partner: Univ. Lund, ILL, FZJ-Richter
Sci. Adv. 2016, 2:e1601432


Protein diffusion is pivotal for the functional properties of biological systems as it strongly influences numerous processes such as signal transmission or reactions between proteins. In the dense and crowded environment of the cell cytoplasm, protein contacts are omnipresent and individual proteins will experience interactions with all the surrounding proteins. While attempts have been made previously to use analogies to colloids in order to model crowding effects, the complexity of protein-protein interactions and the need to measure protein diffusion over length scales comparable to the nearest-neighbour distance represent major obstacles for our understanding of the short-time dynamics in these systems. Here we use quasielastic neutron scattering experiments combined with computer simulations to obtain quantitative information about the short-time diffusion of proteins in crowded solutions. We choose two well-characterized and highly stable eye lens proteins, bovine α-crystallin and γB-crystallin, with repulsive and weakly attractive interactions, respectively, and measure their diffusion as a function of concentration. While diffusion slows down with increasing concentration for both proteins, we find a dramatic decrease of its short-time diffusion coefficient for γB-crystallin by almost three orders of magnitude. Supported by computer simulations of colloid-like protein models, we attribute these drastic changes to specific, anisotropic, patchy short-range protein-protein interactions, which ultimately lead to the formation of large-scale temporal structures. The present study thus provides new insight into emergent patterns of proteins triggered by specific, but typical, weak interactions. In particular, it implies that traditional in-vitro experiments used to investigate specific protein interactions, recognition processes, and diffusion of proteins under dilute conditions, have to be considered with great caution when trying to understand processes in living cells, even when combined with classical colloid models to incorporate the effects of interactions.





Fig. 3. Formation of transient clusters due to weak short-range attractions. Snapshots showing the configurations of particles at f = 0.1 with two additional attractive patches. The color code corresponds to the size of the cluster, Nc/N, to which the particle belongs. Here, Nc is the number of particles in a cluster, and N is the total number of particles in the system. Note that clusters are only transient and that the cluster size fluctuates in time. See movies S1 and S2 for the two simulations.


Strong isotope effects on effective interactions and phase behavior in protein solutions in the presence of multivalent ions


M.K.Braun, M.Wolf, O.Matsarskaia, S.Da Vela, F.Roosen-Runge, M.Sztucki, R.Roth, F.Zhang and F.Schreiber;
SoftComp partner: Univ. Tuebingen
J.Phys.Chem.B, in print (2017)


In this article, we have studied the influence of the isotopic composition of the solvent (H2O or D2O) on the effective interactions and the phase behavior of the globular protein bovine serum albumin in solution with two trivalent salts (LaCl3 and YCl3). Protein solutions with both salts exhibit a reentrant condensation phase behavior. For a constant protein concentration three regimes are found when the salt concentration is continuously increased. In regime I and in regime III the solutions are clear. In the condensed regime (regime II), which is located between two salt concentration boundaries (c* < cs < c**), the solutions become turbid due to protein aggregation or liquid-liquid phase separation (LLPS). This regime II is significantly broadened by replacing H2O with D2O. The samples that undergo LLPS have a lower critical solution temperature (LCST). The value of the transition temperature decreases significantly with increasing solvent fraction of D2O. The effective protein-protein interactions characterized by small-angle X-ray scattering demonstrate that although changing the solvent has negligible effects below c*, where the interactions are dominated by electrostatic repulsion, an enhanced effective attraction is observed in D2O above c*, consistent with the phase behavior observed. As the LCST-LLPS is an entropy-driven phase transition, the results of this study emphasize the role of entropy in solvent isotope effects.





Left: Experimental state diagram of the BSA-LaCl3 system at room temperature in H2O (a) and D2O (b). Shaded areas correspond to the condensed regime (regime II).
Right: Reduced 2nd virial coefficients calculated for the BSA-LaCl3 samples. Triangles and diamonds denote samples in H2O. Samples in D2O are denoted by squares. Solid squares indicate samples showing LLPS.


Kinetics of liquid-liquid phase separation in protein solutions exhibiting LCST phase behavior studied by time-resolved USAXS and VSANS


S. Da Vela, M. K. Braun, A. Doerr, A. Greco, J. Mueller, Z. Fu, F. Zhang, and F. Schreiber
SoftComp partner: Univ. Tuebingen
Soft Matter 12.46, 9334-9341 (2016)


Phase separation in protein solutions is a topic relevant to the colloidal stability and formulation of biopharmaceuticals. In particular, considerable interest has been shown in the last few years in arrested phase separation. Dinamycally arrested states, such as gels or glasses, can be obtained in protein solutions by arrested spinodal decomposition as a consequence of the interplay of a liquid-liquid phase separation (LLPS) binodal with a glass line. LLPS occurs in protein solutions featuring short range attractive interactions, and its phase boundaries can feature upper or lower critical solution temperature (UCST or LCST). In this work, we address the kinetics of LLPS and of arrested LLPS in a solution of bovine serum albumin (BSA) in the presence of the trivalent cation Y(III), which was shown previously to induce a LCST-LLPS behavior in BSA solutions. The techniques of choice are time resolved ultra small-angle X-ray scattering (USAXS) and very small-angle neutron scattering (VSANS). The measurements were performed at beamline ID2 of the ESRF (Grenoble, France) and at beamline KWS3 of the MLZ (Garching, Germany). The combination of the two techniques allows to follow the phase transition from the sub-second regime up to 10^4 s, after performing a temperature jump in the two-phase region of the phase diagram. The scattering profiles feature the characteristic peak associated with spinodal decomposition, growing in intensity and shifting to lower values of q. The growth of the characteristic length scale of the two-phase system changes with the final temperature of the temperature jump. Below 45 ∞C, the characteristic length scale increases with time with a power of about 1/3 for different sample compositions. However, above 45 ∞C, the characteristic length scale follows initially the 1/3 power law growth, then undergoes a significant slowdown, and an arrested state is reached below the denaturation temperature of the protein. This growth kinetics may indicate that the final composition of the protein-rich phase is located close to the high density branch of the LLPS binodal when a kinetically arrested state is reached.




Left: Typical USAXS profiles for in the early stage of spinodal decomposition (the interval between the acquisition of each profile is 0.32 s) upon a temperature jump to Tjump = 35 ∞C in log and linear scale.
Right: Characteristic length extracted from the scattering profiles as a function of time, for different final temperatures and fixed composition of the solution. For the highest temperatures the growth is arrested, as the high-density phase reaches a glassy state.


Cation-induced hydration effects cause lower critical solution temperature behavior in protein solutions


O.Matsarskaia, M.K.Braun, F.Roosen-Runge, M.Wolf, F.Zhang, R.Roth and F.Schreiber
SoftComp partner: Univ. Tuebingen
J.Phys.Chem.B 120.31, 7731-7736 (2016)


The phase behavior of protein solutions is important for numerous phenomena in biology and soft matter. We report a lower critical solution temperature (LCST) phase behavior of aqueous solutions of a globular protein induced by multivalent metal ions around physiological temperatures. The LCST behavior manifests itself via a liquid-liquid phase separation of the protein-salt solution upon heating. Isothermal titration calorimetry and zeta-potential measurements indicate that here cation-protein binding is an endothermic, entropy-driven process. We offer a mechanistic explanation of the LCST. First, cations bind to protein surface groups driven by entropy changes of hydration water. Second, the bound cations bridge to other protein molecules, inducing an entropy-driven attraction causing the LCST. Our findings have general implications for condensation, LCST, and hydration behavior of (bio)polymer solutions as well as the understanding of biological effects of (heavy) metal ions and their hydration.




Left: mechanism of LCST behavior. In the unbound state, the cations and the hydrophilic protein sites are hydrated. Upon cation binding and cation bridging between proteins, water molecules are released and the system entropy is increased.
Right: LCST-LLPS coexistence surface calculated for a protein model with cation-activated attractive patches with a binding free energy based on the thermodynamical characterization of the cation binding


Monolayers of hard rods on planar substrates


M.Klopotek, H.Hansen–Goos, M.Dixit, E.Empting, T.Schilling, F.Schreiber and M.Oettel
SoftComp partner: Univ. Tuebingen
Part I. Equilibrium: JCP 145, 074902 (2016) DOI: 10.1063/1.4960618
Part II. Growth: JCP 146, 084903 (2017) DOI: 10.1063/1.4976308


We model purely hard rods at the vicinity of a substrate both in a lattice model, where posi- tion and orientation of rods are restricted, and in a continuum model with hard spherocylinders, where both position and orientation are continuous variables. In a systematic, two–part study combining analytic theory and simulation we have determined the properties of these monolay- ers in equilibrium, as well as in non–equilibrium under conditions of monotonic growth by means of random deposition. Special attention is given to the orientational ordering of the rods: our generic model showcases the “standing–up” transition found in molecular thin film growth, e.g. with organic molecules, which are rod–like in shape, both in experiment and in recent simulations. The transition from ‘lying’ to ‘standing’ is thermodynamically continuous with hard rods both on the lattice and in the continuum. It remains so for a strong, orientation–dependent attractive substrate; however, this introduces spatio–temporal correlations during non–equilibrium growth: the self–assembly becomes sensitive to the diffusion coefficient of the rods at the substrate. De- tails of monolayer growth are relevant for the cases of Frank–van–der–Merwe (layer–by–layer) and Stranski–Krastanov (islands–on–layers) growth: in ongoing simulations of multi–layer growth, we recover the standing–up transition in the monolayer regime before further layers self–assemble. Slow monolayer growth is characterized by dynamic observables approaching a quasi–equilibrium master curve. Both models—lattice and continuum—in fact reveal qualitatively the same ensemble behavior during highly non–equilibrium growth, differing only via their respective equation–of– states. We discuss how mapping dynamic quantities between models means matching microscopic and macroscopic time–scales non–trivially.
Our equilibrium studies employ Monte Carlo (MC) simulations in the Grand Canonical ensemble, classical lattice Density Functional Theory (DFT) for lattice systems, and continuum MC simula- tions; in non–equilibrium, kinetic Monte Carlo, dynamic DFT on the lattice, and dynamic MC for Brownian dynamics in the continuum were employed.




Monolayers of hard rods manifested in two ways: A lattice model (left) and a continuum model with spherocylinders (right). Colors are used to discriminate rod orientations.


Detection and Imaging of Quorum Sensing in Pseudomonas Aeruginosa Biofilm Communities by Surface-Enhanced Resonance Raman Scattering


G.Bodelón, V.Montes-García, V.López-Puente, E.H.Hill, C.Hamon, M.N.Sanz-Ortiz, S.Rodal-Cedeira, C.Costas, S.Celiksoy, I.Pérez-Juste, L.Scarabelli, A.La Porta, J.Pérez-Juste, I.Pastoriza-Santos, L.M.Liz-Marzán
SoftComp partner: CIC biomaGUNE, UVIGO
Nature Materials 15 (2016) 1203-1211
Article: http://www.nature.com/nmat/journal/v15/n11/full/nmat4720.html


Most bacteria in nature exist as biofilms, which support intercellular signalling processes such as quorum sensing (QS), a cell-to-cell communication mechanism that allows bacteria to monitor and respond to cell density and changes in the environment. As QS and biofilms are involved in the ability of bacteria to cause disease, there is a need for the development of methods for the non-invasive analysis of QS in natural bacterial populations. Here, by using surface-enhanced resonance Raman scattering spectroscopy, we report rationally designed nanostructured plasmonic substrates for the in situ, label-free detection of a QS signalling metabolite in growing Pseudomonas aeruginosa biofilms and microcolonies. The in situ, non-invasive plasmonic imaging of QS in biofilms provides a powerful analytical approach for studying intercellular communication on the basis of secreted molecules as signals.



The SERS-based sensing approaches described in this work not only provide the necessary tools to probe important questions in QS, but can also serve as general detection systems to be applied for the investigation of other cellular communication processes based on SERS-active diffusible molecules.


Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases


D.Niether, D.Afanasenkau, J.K.G.Dhont and Simone Wiegand
SoftComp partner: FZJ-Dhont
PNAS, 113(2016) 4272
D.Niether and Simone Wiegand
SoftComp partner: FZJ-Dhont
Entropy, 19(2017) 33


One of the central questions of humankind is: which chemical and physical conditions are necessary to make life possible? In this “origin-of-life” context, formamide plays an important role, because it has been demonstrated that prebiotic molecules can be synthesized from concentrated formamide solutions. Using finite-element calculations combining thermophoresis and convection processes in hydrothermal pores we showed that sufficiently high formamide concentrations can be accumulated to form prebiotic molecules. Depending on the initial formamide concentration, the aspect ratio of the pores, and the ambient temperature, formamide concentrations up to 85 wt % could be reached after 45–90 d, starting with an initial formamide weight fraction of 10−3 wt % that is typical for concentrations in shallow lakes on early Earth. The stationary calculations show an effective accumulation, only if the aspect ratio is above a certain threshold, and the corresponding transient studies display a sudden increase of the accumulation after a certain time. To explain both observations we derived a simple heuristic model. The physical idea of the approach is a comparison of the time to reach the top of the pore with the time to cross from the convective upstream towards the convective downstream. If the time to reach the top of the pore is shorter than the crossing time, the formamide molecules are flushed out of the pore. If the time is long enough, the formamide molecules can reach the downstream and accumulate at the bottom of the pore. Analyzing the optimal aspect ratio as function of concentration, we find that, at a weight fraction of 0.5, a minimal pore height is required for effective accumulation. At the same concentration, the transient calculations show a maximum of the accumulation rate.



Finite element simulations show that a combination of convection and thermodiffusion enriches formamide in the pore (contour plot). The graph illustrates the accumulation normalized to the reservoir concentration ω0 over time for different ω0. The accumulation saturates around a formamide concentration of 85 wt%. The time necessary to reach the plateau τplateau depends on the reservoir concentration ω0 (inset).
Last modified: 02/03/2017