J. Appl. Cryst.の発刊に際して

Liquid–liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates. Elucidating the molecular driving forces underlying liquid–liquid phase separation is a key objective for understanding biological function and malfunction. Here the authors show that a wide range of cellular proteins, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, which form condensates at low salt concentrations, can reenter a phase-separated regime at high salt concentrations.

/pdf/reentrant-liquid-condensate-phase-of-proteins-is-stabilized-8ll19j9vyr.pdf

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

Many cellular proteins have the ability to demix spontaneously from solution to form liquid condensates. These phase-separated structures form membraneless compartments in living cells and have wide-ranging roles in health and disease. Elucidating the molecular driving forces underlying liquid-liquid phase separation (LLPS) of proteins has thus become a key objective for understanding biological function and malfunction. Here we show that proteins implicated in cellular phase separation, such as FUS, TDP-43, and Annexin A11, which form condensates at low salt concentrations via homotypic multivalent interactions, also have the ability to undergo LLPS at high salt concentrations by reentering into a phase-separated regime. Through a combination of experiments and simulations, we demonstrate that phase separation in the high-salt regime is mainly driven by hydrophobic and non-ionic interactions. As such, it is mechanistically distinct from the low-salt regime, where condensates are stabilized by a broad mix of electrostatic, hydrophobic, and non-ionic forces. Our work thus expands the molecular grammar of interactions governing LLPS of cellular proteins and provides a new view on hydrophobicity and non-ionic interactions as non-specific driving forces for the condensation process, with important implications for the aberrant function, druggability, and material properties of biomolecular condensates.

/pdf/reentrant-liquid-condensate-phase-of-proteins-is-stabilized-r5k3oy3ntw.pdf

The use of polyoxometalates in protein crystallography - An attempt to widen a well-known bottleneck.

Better predictive ability of salt and buffer effects on protein-protein interactions requires separating out contributions due to ionic screening, protein charge neutralization by ion binding, and salting-in(out) behavior. We have carried out a systematic study by measuring protein-protein interactions for a monoclonal antibody over an ionic strength range of 25 to 525 mM at 4 pH values (5, 6.5, 8, and 9) in solutions containing sodium chloride, calcium chloride, sodium sulfate, or sodium thiocyante. The salt ions are chosen so as to represent a range of affinities for protein charged and noncharged groups. The results are compared to effects of various buffers including acetate, citrate, phosphate, histidine, succinate, or tris. In low ionic strength solutions, anion binding affinity is reflected by the ability to reduce protein-protein repulsion, which follows the order thiocyanate > sulfate > chloride. The sulfate specific effect is screened at the same ionic strength required to screen the pH dependence of protein-protein interactions indicating sulfate binding only neutralizes protein charged groups. Thiocyanate specific effects occur over a larger ionic strength range reflecting adsorption to charged and noncharged regions of the protein. The latter leads to salting-in behavior and, at low pH, a nonmonotonic interaction profile with respect to sodium thiocyanate concentration. The effects of thiocyanate can not be rationalized in terms of only neutralizing double layer forces indicating the presence of an additional short-ranged protein-protein attraction at moderate ionic strength. Conversely, buffer specific effects can be explained through a charge neutralization mechanism, where buffers with greater valency are more effective at reducing double layer forces at low pH. Citrate binding at pH 6.5 leads to protein charge inversion and the formation of attractive electrostatic interactions. Throughout the report, we highlight similarities in the measured protein-protein interaction profiles with previous studies of globular proteins and of antibodies providing evidence that the behavior will be common to other protein systems.

Specific Ion and Buffer Effects on Protein–Protein Interactions of a Monoclonal Antibody

The effective interactions and phase behavior of protein solutions under strong electrostatic coupling conditions are difficult to understand due to the complex charge pattern and irregular geometry of protein surfaces. This distinguishes them from related systems such as DNA or conventional colloids. In this work, we discuss the question of universality of the reentrant condensation (RC) of proteins in solution induced by multivalent counterions, i.e., redissolution on adding further salts after phase separation, as recently discovered (Zhang et al., Phys Rev Lett 2008; 101:148101). The discussion is based on a systematic investigation of five different proteins with different charge patterns and five different multivalent counterions. Zeta potential measurements confirm the effective charge inversion of proteins in the reentrant regime via binding of multivalent counterions, which is supported by Monte Carlo simulations. Charge inversion by trivalent cations requires an overall negative net charge of the protein. Statistical analysis of a representative set of protein sequences reveals that, in theory, this effect could be possible for about half of all proteins. Our results can be exploited for the control of the phase behavior of proteins, in particular facilitating protein crystallization. Proteins 2010. © 2010 Wiley-Liss, Inc.

Universality of protein reentrant condensation in solution induced by multivalent metal ions.

Tuning of protein surface charge is a fundamental mechanism in biological systems. Protein charge is regulated in a physiological context by pH and interaction with counterions. We report on charge inversion and the related reentrant condensation in solutions of globular proteins with different multivalent metal cations. In particular, we focus on the changes in phase behavior and charge regulation due to pH effects caused by hydrolysis of metal ions. For several proteins and metal salts, charge inversion as measured by electrophoretic light scattering is found to be a universal phenomenon, the extent of which is dependent on the specific protein-salt combination. Reentrant phase diagrams show a much narrower phase-separated regime for acidic salts such as AlCl3 and FeCl3 compared to neutral salts such as YCl3 or LaCl3 . The differences between acidic and neutral salts can be explained by the interplay of pH effects and binding of the multivalent counterions. The experimental findings are reproduced with good agreement by an analytical model for protein charging taking into account ion condensation, metal ion hydrolysis and interaction with charged amino acid side chains on the protein surface. Finally, the relationship of charge inversion and reentrant condensation is discussed, suggesting that pH variation in combination with multivalent cations provides control over both attractive and repulsive interactions between proteins.

Interplay of pH and Binding of Multivalent Metal Ions: Charge Inversion and Reentrant Condensation in Protein Solutions

Tuning of protein surface charge is a fundamental mechanism in biological systems. Protein charge is regulated in a physiological context by pH and interaction with counterions. We report on charge inversion and the related reentrant condensation in solutions of globular proteins with different multivalent metal cations. In particular, we focus on the changes in phase behavior and charge regulation due to pH effects caused by hydrolysis of metal ions. For several proteins and metal salts, charge inversion as measured by electrophoretic light scattering is found to be a universal phenomenon, the extent of which is dependent on the specific protein-salt combination. Reentrant phase diagrams show a much narrower phase-separated regime for acidic salts such as AlCl3 and FeCl3 compared to neutral salts such as YCl3 or LaCl3. The differences between acidic and neutral salts can be explained by the interplay of pH effects and binding of the multivalent counterions. The experimental findings are reproduced with good agreement by an analytical model for protein charging taking into account ion condensation, metal ion hydrolysis, and interaction with charged amino acid side chains on the protein surface. Finally, the relationship of charge inversion and reentrant condensation is discussed, suggesting that pH variation in combination with multivalent cations provides control over both attractive and repulsive interactions between proteins.

http://www.soft-matter.uni-tuebingen.de/publications/Roosen-Runge_jpcb13.pdf

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Universality of protein reentrant condensation in solution induced by multivalent metal ions.

Interplay of pH and Binding of Multivalent Metal Ions: Charge Inversion and Reentrant Condensation in Protein Solutions

Interplay of pH and Binding of Multivalent Metal Ions: Charge Inversion and Reentrant Condensation in Protein Solutions