The role of cluster formation and metastable liquid-liquid phase separation in protein crystallization

We discuss the phase behavior and in particular crystallization of a model globular protein (beta-lactoglobulin) in solution in the presence of multivalent electrolytes. It has been shown previously that negatively charged globular proteins at neutral pH in the presence of multivalent counterions un...

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Bibliographic Details
Published in:Faraday discussions 2012-01, Vol.159, p.313-325
Main Authors: Zhang, Fajun, Roosen-Runge, Felix, Sauter, Andrea, Roth, Roland, Skoda, Maximilian W. A, Jacobs, Robert M. J, Sztucki, Michael, Schreiber, Frank
Format: Article
Language:English
Subjects:
Clusters
Crystal growth
Crystallization
Crystals
Liquid phases
Nucleation
Phase separation
Proteins
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Summary:We discuss the phase behavior and in particular crystallization of a model globular protein (beta-lactoglobulin) in solution in the presence of multivalent electrolytes. It has been shown previously that negatively charged globular proteins at neutral pH in the presence of multivalent counterions undergo a "re-entrant condensation (RC)" phase behavior (Zhang et al. , Phys. Rev. Lett. , 2008, 101 , 148101), i.e. a phase-separated regime occurs in between two critical salt concentrations, c * < c **, giving a metastable liquid-liquid phase separation (LLPS). Crystallization from the condensed regime has been observed to follow different mechanisms. Near c *, crystals grow following a classic nucleation and growth mechanism; near c **, the crystallization follows a two-step crystallization mechanism, i.e , crystal growth follows a metastable LLPS. In this paper, we focus on the two-step crystal growth near c **. SAXS measurements indicate that proteins form clusters in this regime and the cluster size increases approaching c **. Upon lowering the temperature, in situ SAXS studies indicate that the clusters can directly form both a dense liquid phase and protein crystals. During the crystal growth, the metastable dense liquid phase is dissolved. Based on our observations, we discuss a nucleation mechanism starting from clusters in the dilute phase from a metastable LLPS. These protein clusters behave as the building blocks for nucleation, while the dense phase acts as a reservoir ensuring constant protein concentration in the dilute phase during crystal growth.
ISSN:1359-6640
1364-5498
DOI:10.1039/c2fd20021j