Influence of the Roughness, Topography, and Physicochemical Properties of Chemically Modified Surfaces on the Heterogeneous Nucleation of Protein Crystals

Abstract

In this study, the influence of some factors on the heterogeneous nucleation of hen egg-white lysozyme (E.C. 3.2.1.17) on a series of chemically modified surfaces has been investigated. Microbatch crystallization experiments were conducted on the microscope glass slides that were treated with poly-L-glutamic acid (PLG), poly(2-hydroxyethyl methacrylate) (P2HEMA), poly(methyl methacrylate) (PMMA), poly(4-vinyl pyridine) (P4VP), and (3-aminopropyl)triethoxysilane (APTES). An optical microscope with a heating/cooling stage was employed to measure the induction time of heterogeneous nucleation. The surface topography and roughness were characterized by atomic force microscopy. Contact angles for crystallization solution on the investigated surfaces were measured by a contact angle meter. From the theoretical analysis, the energetic barrier to heterogeneous nucleation was found to increase at higher contact angles and to decrease at higher roughness. Experimentally, a qualitative increase of the induction time of the heterogeneous nucleation on P2HEMA, APTES, and PMMA surfaces with the contact angle was observed. Such surfaces as P2HEMA, PLG, and APTES, which were of higher roughness, were shown to promote the heterogeneous nucleation. In addition, the surface with specific topography is expected to increase the possibility of the formation of a critical nucleus. Finally, the P4VP surface appeared to suppress the heterogeneous nucleation as a result of the electrostatic interaction between the lysozyme and P4VP molecules.

Enhancement of nucleation during hanging drop protein crystallization using HF Treatment of cover glasses

“…At the etched surface, the protein will thus be more concentrated, resulting in a higher regional supersaturation, which is favorable for crystal nucleation. This is a widely accepted reason that nucleation can occur near a roughened surface at a metastable zone rather than at the nucleation zone in bulk solution [20,21]. 2) The larger contact area provides more opportunities for nucleation.…”

“…Etched surfaces provide heterogeneous nucleation sites so that crystallization can occur at lower supersaturation conditions, thus better quality crystals can be obtained. In this paper, we did not assess the quality of the crystals obtained at lower supersaturation conditions using modified surfaces, but in the literature, related references can be found [21]. 3) Application in automated crystallization In the field of protein crystallization, automated crystallization screens using liquid handling robots has become a trend, and more and more laboratories will take advantage of this system in practical crystallization screens and optimization.…”

Protein Crystallization by Anodic Porous Alumina (APA) Template: The Example of Hen Egg White Lysozyme (HEWL)

“…In order to control heterogeneous nucleation of lysozyme crystals, hair cuticles [8] or ad hoc engineered structured surfaces such as Poly-L-Lysine modified glass substrate [9], chemically modified mica surfaces [10] or other chemically modified patterns [11], xanthenes dyes [12], polystyrene nanospheres [13], porous glass [14], porous silicon [15] and fluorinated layered silicate (which is a phyllosilicate with a parallel two-dimensional lamellar structure; Ino et al, 2011) [16] can be exploited. In the last work, the fluorine atoms were considered responsible for driving the nucleation process.…”

Polymorphism of pharmaceutical molecules: perspectives on nucleation

“…Nucleation will happen via the compact critical cluster comprising molecules forming suitable bond angles with their neighbors. This means that the hydrophobicity and pore size will result in the selectivity of the heterogeneous nucleation [37].…”