Direct and Noninvasive Observation of Two-Dimensional Nucleation Behavior of Protein Crystals by Advanced Optical Microscopy

Abstract

We observed two-dimensional (2D) nucleation behavior on {110} and {101} faces of tetragonal crystals of model protein lysozyme by laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM). We measured, for the first time directly and noninvasively, the 2D nucleation rates using 99.99% pure lysozyme, 98.5% pure lysozyme (Seikagaku Co.), and 99.99% pure lysozyme with intentionally added impure proteins (fluorescent-labeled lysozyme, covalently bonded dimer of lysozyme, and 18 kDa polypeptide). We found that 2D nucleation was the dominant growth mechanism under conditions adopted in this study, and the 2D nucleation occurred randomly on the entire crystal surface irrespective of supersaturation within the range of σ ) ln(C/C e ) ) 0-1.4, where C is a bulk lysozyme concentration and C e the solubility (crystal size: 0.2-0.3 mm). Repeated 2D nucleation, which continued for 3-4 layers, was also observed mainly when the impure proteins were present. In addition, multilayered 2D islands were formed after the adsorption of relatively large foreign particles on the crystal surface. From the comparison between the 2D nucleation rates determined on the {110} faces with and without the impure proteins, we concluded that homogeneous 2D nucleation occurred under a higher supersaturation range (σ > 0.8), irrespective of the presence of the impurities. In contrast, under a lower supersaturation range (σ < 0.8), we found that significant heterogeneous 2D nucleation dominated the growth mainly when the impure proteins were present. The {101} faces exhibited more significant heterogeneous 2D nucleation induced by smaller amounts of impurities than in the case of the {110} faces. We also determined the ledge free energies of the homogeneous and heterogeneous nucleation. Within the experimental conditions used in this study, we could not find significant dependence of the ledge free energies of the heterogeneous nucleation on the kinds of impure proteins.

Role of clusters in nonclassical nucleation and growth of protein crystals

“…S1 and Movie S1. These looped macrosteps are different from other macrostepped features observed on protein crystals in that they are not the result of a complex growth mechanism [arising from, e.g., step bunching due to diffusion field overlap, solutal flow, Schwoebel effect, impurities, repeated 2D nucleation (24)] but rather nucleate seemingly unprompted enclosing a single elevated area on the surface. Subsequent lateral growth then yields an expanding mound that contains a considerable amount of steps (ranging from 2 to >100).…”

Effect of an External Electric Field on the Kinetics of Dislocation-Free Growth of Tetragonal Hen Egg White Lysozyme Crystals

“…A dislocation-free crystal cross-linked for 15 min [58] was used in this experiment, so that 2D nucleation and growth of the birth-and-spread type should only occur on the growing faces. Under the range of supersaturations used in this experiment, the formation of multiple nuclei at several points on one growing face (multiple nucleation mode) has been observed for tetragonal HEW lysozyme crystals [62]. The growth rate R using the birth-and-spread model is expressed as [63,64]:…”

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

“…AFM is able merely to visualize elementary acts during protein and virus crystal growth [59,60], while the sizes of the protein crystal nuclei are determined by means of thermodynamic estimations. Using LCM-DIM, Sazaki's group studies the 2-D nucleation kinetics of lysozyme [61] and glucose isomerase crystals (under high pressure) [62]. However, the main difficulty in the experimental study of the crystal nucleation arises out of the fact that it is impossible to distinguish the critical nuclei in the whole assembly of under-critical, critical and super-critical molecular clusters; cluster composition changes dynamically due to the constant growth/decay of differently sized clusters; and critical nuclei are not labeled.…”