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Volume 159, 2012
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The role of cluster formation and metastable liquid—liquid phase separation in protein crystallization

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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.

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Publication details

The article was received on 15 Feb 2012, accepted on 19 Mar 2012 and first published on 20 Mar 2012

Article type: Paper
DOI: 10.1039/C2FD20021J
Citation: Faraday Discuss., 2012,159, 313-325
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    The role of cluster formation and metastable liquid—liquid phase separation in protein crystallization

    F. Zhang, F. Roosen-Runge, A. Sauter, R. Roth, M. W. A. Skoda, R. M. J. Jacobs, M. Sztucki and F. Schreiber, Faraday Discuss., 2012, 159, 313
    DOI: 10.1039/C2FD20021J

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