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

Zhang, Fajun ; Roosen-Runge, F. LU ; Sauter, Andrea ; Roth, Roland ; Skoda, Maximilian W.A. ; Jacobs, Robert M.J. ; Sztucki, Michael and Schreiber, Frank (2012) In Faraday Discussions 159. p.313-325
Abstract

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... (More)

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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author
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publishing date
type
Contribution to journal
publication status
published
in
Faraday Discussions
volume
159
pages
13 pages
publisher
Royal Society of Chemistry
external identifiers
  • scopus:84867952155
ISSN
1359-6640
DOI
10.1039/c2fd20021j
language
English
LU publication?
no
id
619a2d1c-5c2d-41c1-816b-d16123917e39
date added to LUP
2018-12-17 09:48:36
date last changed
2022-04-18 01:09:01
@article{619a2d1c-5c2d-41c1-816b-d16123917e39,
  abstract     = {{<p>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* &lt; 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.</p>}},
  author       = {{Zhang, Fajun and Roosen-Runge, F. and Sauter, Andrea and Roth, Roland and Skoda, Maximilian W.A. and Jacobs, Robert M.J. and Sztucki, Michael and Schreiber, Frank}},
  issn         = {{1359-6640}},
  language     = {{eng}},
  month        = {{11}},
  pages        = {{313--325}},
  publisher    = {{Royal Society of Chemistry}},
  series       = {{Faraday Discussions}},
  title        = {{The role of cluster formation and metastable liquid - Liquid phase separation in protein crystallization}},
  url          = {{http://dx.doi.org/10.1039/c2fd20021j}},
  doi          = {{10.1039/c2fd20021j}},
  volume       = {{159}},
  year         = {{2012}},
}