Liquid-Liquid Phase Separation in Protein Solutions Controlled by Multivalent Salts and Temperature Felix Roosen-Runge 1 , Christodoulos Christodoulou 1 , Fajun Zhang 1 , Marcell Wolf 1 , Roland Roth 2 and Frank Schreiber 1 1 Institut f ¨ ur Angewandte Physik, Universit¨ at T ¨ ubingen, Germany 2 Institut f ¨ ur Theoretische Physik I, Universit¨ at Erlangen-N ¨ urnberg, Germany Abstract Solutions of most globular proteins are charge-stabilized. Charge screening, salting-in and salting-out behavior via addition of salts are available ways controlling the phase behavior of the solutions. However, multivalent salts have been found to induce even more complex effects in protein solutions. In particular, reentrant condensation is observed for several globular proteins in the presence of multivalent salts (1, 2), i.e. the protein solution is stable at low and high salt concentration but phase-separates at intermediate salt concentration. As conclusive explanation, specific ion binding to the protein residues and pH effects cause an inversion of the protein global charge. Importantly, besides the expected amorphous aggregation at low charge-stabilization, also a liquid-liquid phase separation occurs for several proteins at intermediate salt concentration. The full phase diagram of the protein solution extends to three control parameter – temperature, protein and salt concentration. Considering the protein-ion complexes as effective particles with short-range attractions whose strength varies with the protein charge, the full phase behavior can be explained. Interestingly, the observed lower critical solution point suggests an inportant role of hydration to the free energy of the protein solution. Reentrant condensation and liquid-liquid phase separation (LLPS) (1-3) for given protein concentration c p with increasing multivalent salt concentration c s : clear solution  amorphous aggregation and / or (metastable liquid-liquid) phase separation  clear solution universal phenomenon for • globular proteins with initial negative charge (e.g. BSA, HSA, β -LG) and • several multivalent salts (e.g. FeCl 3 , YCl 3 ) from UVvis and X-ray absorption: • LLPS occurs in closed area within the condensation regime Charge inversion upon addition of multivalent ions (4) • surface charge Q via electrophoresis • binding isotherm: Q = Q 0 + ZNc s c s + K ′ exp(pQ ) • additional pH effect from hydrolysis of mulivalent salts → surface charge inversion due to pH effects and binding of multivalent counterions Lower critical solution temperature (LCST) • upon heating the protein-salt solution: absorption (i.e. turbidity) sharply increases at the LCST → LCST implies significant entropic contributions, e.g. hydration effects Binodal and spinodal lines Binodal line • phase separation at defined temperature • centrifugation to seperate phases • protein concentration for dilute phase (UVvis) • dense phase very viscous → gelation? Spinodal line • divergence of scattering at spinodal point → determine via static light scattering Interpretation: threedimensional phase diagram (3) L-L F-S S F 1/T c s c p protein-ion complexes have • different tunable interactions • phase diagram with metastable LLPS (as known for colloids with short-range attraction) Outlook • experimentally: broader range of conditions: mechanism of phase separation and gelation, effect of hydration • theoretically: conceptual frame connecting the phenomena • technologically: control mechanism of phase behavior of protein solutions, protein crystallization Acknowledgements • Deutsche Forschungsgemein- schaft, Germany: funding • Studienstiftung des deutschen Volkes, Germany: scholarship • ESRF, Grenoble, France: beamtime allocation Literature (1) F. Zhang et al., Reentrant Condensation of Proteins in Solution Induced by Multivalent Counterions, PRL 101, 148101 (2008) (2) F. Zhang et al., Universality of protein reentrant condensation in solution induced by multivalent metal ions, Proteins 78(16), 3450–3457 (2010) (3) F. Zhang et al., Metastable Liquid-Liquid Phase Separation in Protein Solutions as a Universal Pathway Towards Crystallization, submitted (2011) (4) F. Roosen-Runge et al., Inversion of Protein Surface Charge Caused by Binding of Multivalent Ions, in preparation http://www.soft-matter.uni-tuebingen.de