Abstract
We report a real-time study on protein crystallization in the presence of multivalent salts using small angle X-ray scattering (SAXS) and optical microscopy, focusing particularly on the nucleation mechanism as well as on the role of the metastable intermediate phase (MIP). Using bovine beta-lactoglobulin as a model system in the presence of the divalent salt CdCl2, we have monitored the early stage of crystallization kinetics which demonstrates a two-step nucleation mechanism: protein aggregates form a MIP, which is followed by the nucleation of crystals within the MIP. Here we focus on characterizing and tuning the structure of the MIP using salt and the related effects on the two-step nucleation kinetics. The results suggest that increasing the salt concentration near the transition zone pseudo-c** enhances the energy barrier for both MIPs and crystal nucleation, leading to slow growth. The structural evolution of the MIP and its effect on subsequent nucleation is discussed based on the growth kinetics. The observed kinetics can be well described, using a rate-equation model based on a clear physical two-step picture. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization, but also elucidates the role and the structural signature of the MIPs in the nonclassical process of protein crystallization.
Highlights
We have investigated the two-step nucleation process of protein crystallization in solutions by following the overall crystallization kinetics, using real-time optical microscopy and small angle X-ray scattering (SAXS)
These protein aggregates serve as the metastable intermediate phase (MIP) during crystallization
SAXS and SANS reveal that the MIP shows a certain local ordering instead of random aggregates, as monitored by a broad shoulder at intermediate q z 0.7 nmÀ1, and a monomer–monomer correlation peak at q around 2 nmÀ1
Summary
Paper that a metastable intermediate phase (MIP) exists before the nal crystal structure is formed,[7,8,9,10,11,12,13,14,15,16,17,18,19,20,21] i.e. the solutes in the supersaturated solution form, in a rst step, either small clusters or a macroscopic dense liquid phase. Colloidal systems exhibit similar phase behavior to atomic and molecular systems, and their large particle sizes enable visualization on a single-particle level Using this technique, Tan et al studied the liquid– solid phase transition and observed the formation of a metastable precursor under their experimental conditions, regardless of the nal state and the interaction potential.[24] Peng et al studied the kinetics of a solid–solid phase transition using single-particle resolution video microscopy. Tan et al studied the liquid– solid phase transition and observed the formation of a metastable precursor under their experimental conditions, regardless of the nal state and the interaction potential.[24] Peng et al studied the kinetics of a solid–solid phase transition using single-particle resolution video microscopy They observed that the transition between two different solid states occurs via a two-step diffusive nucleation pathway involving liquid nuclei.[25] This pathway is favored in comparison to onestep nucleation, because the energy of the solid–liquid interface is lower than that between the solid phases. We focus on the effect of the structural property of the MIP on the nucleation and growth of the two-step nucleation mechanism
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