Two types of amorphous protein particles facilitate crystal nucleation

Abstract

Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used timeresolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.nucleation | protein | lysozyme | transmission electron microscopy | in situ observation C rystallization can be divided into two processes: nucleation and crystal growth. The crystal growth process has been well examined for a long time, yet the nucleation process is not understood; for example, the nucleation rate of crystals provides a textbook example of order-of-magnitude discrepancies between theoretical predictions and experimental results. Recent proposals have attributed these discrepancies to a nonclassical nucleation pathway, along which a structured crystalline embryo forms within a highly concentrated disordered precursor (1). This mechanism was first proposed for protein crystals (2, 3). Direct observations have demonstrated its applicability to organic (4), inorganic (5, 6), and colloidal (7) crystals. In proteins, clusters of protein molecules have been suggested as precursors; these clusters have mesoscopic sizes from several tens to several hundreds of nanometers and are considered to behave like liquids. It has also been suggested that the precursor is thermodynamically stable with respect to the mother liquid phase but is metastable or unstable with respect to the crystalline phase. The latter nature of the precursor differs from the stable macroscopically dense liquid formed as a result of the liquid-liquid phase separation (8). Such protein-rich mesoscopic clusters have been observed for many proteins, primarily using optical techniques, and have been tentatively identified as precursors for crystal nucleation (9-12). Several important questions concerning this mechanism remain unanswered. First, are the observed mesoscopic clusters actually liquid-like or solid-amorphous? Second, do they play an active role in crystal nucleation? In addition, finally, do the clusters serve as classical heteroge...

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

“…This has been concluded by Yamazaki et al [56] (who conducted experiments with LC-TEM). The authors have established that mesoscopic clusters, similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are not liquid but amorphous solid particles consisting of lysozyme molecules.…”

“…For instance, Yamazaki et al [56] have used LC-TEM to understand the mechanisms underlying the early stages of protein crystallization (see Section 2.1).…”

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

“…All experimental data for the dimensionless supersaturations studied (numbers on right-hand side) fall on the orange logistic curves (for the color references, refer to the web version of this article). The dashed straight lines with coordinates (00), (11) are a guide for the eye only; it is seen that the experimental points less than t/t p = 0.5 are situated below the dashed straight line, while the points for greater t/t p values (up to 1) lie above this line. Experimental data are plotted for: (a) bulk nucleation; (b) on-glass nucleation.…”

“…Thus, the relative efficiencies of the various pathways leading towards the final crystalline state have been studied. Most recently, direct transition electron microscopic observations of Yamazaki et al [11] have suggested a significant departure from the initial TSNM assumption. The authors have never observed formation of crystalline phases inside amorphous solid particles consisting of lysozyme molecules, which are like those previously assumed to consist of a dense liquid.…”

Weakly-bound Dimers that Underlie the Crystal Nucleation Precursors in Lysozyme Solutions

“…Surprisingly, recent experimental measurements of nucleation rates revealed that they are even lower, by many orders of magnitude, than those predicted by theory (19)(20)(21). The issue of low nucleation rates and several other unexplained features of protein nucleation kinetics was resolved by the discovery that the nuclei of ordered protein solids form not in the supersaturated solution but rather in preexisting mesoscopic protein-rich clusters (22)(23)(24)(25)(26)(27)(28)(29)(30). In addition to crystals, the mesoscopic clusters host the nucleation of other ordered protein solids, such as sickle-cell hemoglobin polymers (27) and amyloid fibrils (28)(29)(30).…”