Nucleation precursors in protein crystallization

Abstract

Protein crystal nucleation is a central problem in biological crystallography and other areas of science, technology and medicine. Recent studies have demonstrated that protein crystal nuclei form within crucial precursors. Here, methods of detection and characterization of the precursors are reviewed: dynamic light scattering, atomic force microscopy and Brownian microscopy. Data for several proteins provided by these methods have demonstrated that the nucleation precursors are clusters consisting of protein-dense liquid, which are metastable with respect to the host protein solution. The clusters are several hundred nanometres in size, the cluster population occupies from 10(-7) to 10(-3) of the solution volume, and their properties in solutions supersaturated with respect to crystals are similar to those in homogeneous, i.e. undersaturated, solutions. The clusters exist owing to the conformation flexibility of the protein molecules, leading to exposure of hydrophobic surfaces and enhanced intermolecular binding. These results indicate that protein conformational flexibility might be the mechanism behind the metastable mesoscopic clusters and crystal nucleation. Investigations of the cluster properties are still in their infancy. Results on direct imaging of cluster behaviors and characterization of cluster mechanisms with a variety of proteins will soon lead to major breakthroughs in protein biophysics.

Keywords: crystallization; nucleation mechanism; partial unfolding; protein crystals; protein-rich clusters; two-step nucleation.

Figure 1

The two-step mechanism of nucleation of crystals: (i) a metastable cluster forms; (ii) a crystal nucleus may form inside the cluster. (a) Macroscopic viewpoint; the numbers denote the steps in the nucleation mechanism; after nucleation, a crystal irreversibly grows to macroscopic dimensions. (b) Microscopic viewpoint in the (concentration, structure) plane; the thick dashed line highlights the two-step pathway and the diagonal solid arrow highlights direct nucleation. (c) The free energy ΔG along three possible nucleation pathways: direct nucleation, the two-step mechanism and crystals forming within macroscopic dense liquid, as seen by Vivarès et al. (2005 ▶), following the Ostwald rule of stages (Ostwald, 1897 ▶).

Figure 2

Direct imaging of clusters in a lumazine synthase solution by atomic force microscopy. (a, b) Sedimentation of a cluster and its development into a stack of five crystalline layers. Tapping-mode AFM imaging, scan size 20 × 20 µm; the time interval between (a) and (b) is 9 min. (c, d) Height profiles along a horizontal line crossing the three-dimensional object in (a) and (b), respectively, show an object height of ∼120 nm immediately after sedimentation in (a) and of ∼75 nm in (b). The arrows in (a), (b), (c) and (d) mark the same crystal layer. Reprinted with permission from Gliko et al. (2005 ▶), J. Am. Chem. Soc. 127, 3433–3438. Copyright 2005 American Chemical Society.

Figure 3

Examples of the correlation function of the scattered light g ₂(τ) and the intensity distribution function G(τ) of a hemoglobin S solution. The characteristic diffusion times τ₁ and τ₂ and the amplitudes A ₁ and A ₂ of the monomers and clusters, respectively, are indicated. Reprinted from Pan et al. (2007 ▶), with permission from Elsevier.

Figure 4

Brownian microscopy characterization of clusters in a lysozyme solution. (a) Schematic of the cuvette, the illuminating laser beam and the formation of a hologram of a cluster. (b) The clusters, which scatter light much strongly than the monomers, are seen as bright spots. (c) Brownian trajectory of a cluster. (d) The relation between the mean-squared displacement 〈Δr ²〉 and elapsed time Δt, from which the diffusion coefficient and size of the cluster are determined. (e) Characterization of the cluster population in a lysozyme solution by Brownian microscopy. Three independent determinations of the distribution of cluster size. Reprinted with permission from Li et al. (2011 ▶), Rev. Sci. Instrum. 82, 053106. Copyright 2011, AIP Publishing LLC.

Figure 5

Characterization of clusters by dynamic light scattering. (a) Schematic illustration of the motion of clusters suspended in a solution; the clusters and monomers exhibit two distinct diffusion times. (b) Schematic illustration of the motion of molecules embedded in a loose network. The diffusion of the embedded molecules is slower than that for the free molecules in the network voids, resulting in two diffusion times. (c) The dependence of the decay rate of the intensity scattered from clusters in a lysozyme solution Γ₂ on the scattering vector q. (d) The evolution of the mean cluster radius R ₂ during 200 min immediately after solution preparation.

Figure 6

The clusters and the phase diagram of the protein solution. Experimentally determined phase diagram of a lysozyme solution in 0.05 M sodium acetate buffer pH 4.5 and 4.0% NaCl. Liquidus, or solubility, from Cacioppo & Pusey (1991 ▶) and Howard et al. (1988 ▶); liquid–liquid (L–L) coexistence and respective spinodal from Petsev et al. (2003 ▶); solution–crystal spinodal from Filobelo et al. (2005 ▶); gelation line from Petsev et al. (2003 ▶) and Muschol & Rosenberger (1997 ▶). The shaded area denotes compositions at which dense liquid clusters were detected.

Figure 7

Evaluation of the free-energy density of the protein solution. Debye plot of the ratio M _w KC/R _θ as a function of protein mass concentration C. M _w = 14 300 g mol⁻¹ is the molecular mass of lysozyme, K is the instrument constant and R _θ = I _θ/I ₀ is the Raleigh ratio of the intensity scattered at angle θ to the incident. Solid symbols, determination using static light scattering. Dashed line, fit of osmotic virial expansion to data. Solid line, integration of data according to (8) to determine the solution free energy ΔG. Reprinted with permission from Pan et al. (2010 ▶), J. Phys. Chem. B, 114, 7620–7630. Copyright 2010 American Chemical Society.

Figure 8

The mechanism of cluster formation. (a) Concentration profiles in a cluster. n _L, total protein concentration in solution; n _H, total protein concentration in dense liquid inside clusters; r, distance from the center of the cluster; R, cluster radius. Reprinted with permission from Pan et al. (2010 ▶), J. Phys. Chem. B, 114, 7620–7630. Copyright 2010 American Chemical Society. (b) The free-energy variation along the reaction coordinate for formation of the anomalous mesoscopic clusters. Insets: schematic illustrations of the three steps of the cluster-formation mechanism.

Figure 9

The bonds in the transient oligomers. (a) Tests for the role of specific chemical bonds in cluster formation. The evolution of the cluster radius R ₂ in a lysozyme solution as a result of bubbling with helium or air. (b) Schematic illustration of the domain-swapping mechanism of formation of protein oligomers. (c) The effect of urea on cluster formation. Correlation functions from lysozyme solutions at pH 7.8 in 20 mM HEPES buffer collected in the absence and presence of urea, as indicated in the plot. Reprinted with permission from Pan et al. (2010 ▶), J. Phys. Chem. B, 114, 7620–7630. Copyright 2010 American Chemical Society.