Real-Time Observation of Nonclassical Protein Crystallization Kinetics

Abstract

We present a real-time study of protein crystallization of bovine β-lactoglobulin in the presence of CdCl(2) using small-angle X-ray scattering and optical microscopy. From observing the crystallization kinetics, we propose the following multistep crystallization mechanism that is consistent with our data. In the first step, an intermediate phase is formed, followed by the nucleation of crystals within the intermediate phase. During this period, the number of crystals increases with time, but the crystal growth is slowed down by the surrounding dense intermediate phase due to the low mobility. In the next step, the intermediate phase is consumed by nucleation and slow growth, and the crystals are exposed to the dilute phase. In this stage, the number of crystals becomes nearly constant, whereas the crystals grow rapidly due to access to the free protein molecules in the dilute phase. 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 metastable intermediate phase in this process.

Nanostructure formation via post growth of particles

“…However, Ostwald ripening of α-phase would result in two possible outcomes: one is a β-phase product with a larger size but a very narrow size distribution, and other is smaller particles of the rare earth trifluoride REF 3 . However, this kind of dissolution and regrowth method could be utilized to prepare uniform NPs with a narrow size distribution, too.…”

Phenomenological Consideration of Protein Crystal Nucleation; the Physics and Biochemistry behind the Phenomenon

“…Such time dependence has also been established for bovine β-lactoglobulin crystal nucleation which proceeds by a TSNM [6]. Good logistic fits of insulin crystal nucleation data for seven different supersaturations are shown in Figure 3, where appropriate (supersaturation dependent) parameters are used.…”

“…Thus, TSNM resembles the Ostwald's rule of stages, which stipulates that a thermodynamically less-stable phase appears first, then a polymorphic transition toward a stable phase occurs. Ten Wolde and Frenkel [4] have predicted theoretically the existence of amorphous precursors that have been further confirmed experimentally by Vivares et al [5], Sauter et al [6], and Schubert et al [7]. Sleutel and Van Driessche [8] have observed a non-classical nucleation for the 3D liquid-to-crystal transition of glucose isomerase-local increase in density and crystallinity do not occur simultaneously, but rather sequentially.…”

Recent Insights into the Crystallization Process; Protein Crystal Nucleation and Growth Peculiarities; Processes in the Presence of Electric Fields

“…The existence of amorphous nuclei precursors has been confirmed experimentally by Vivares et al [69], Sauter et al [70], and further by Schubert et al [71]. Sleutel and Van Driessche [72] have observed a non-classical nucleation for the 3D liquid-to-crystal transition of glucose isomerase; local increase in density and crystallinity do not occur simultaneously, but rather sequentially.…”