Hydrophobic Interface-Assisted Protein Crystallization: Theory and Experiment

Abstract

Macromolecular crystallization is crucial to a large number of scientific fields, including structural biology; drug design, formulation, and delivery; manufacture of biomaterials; and preparation of foodstuffs. The purpose of this study is to facilitate control of crystallization, by investigating hydrophobic interface-assisted protein crystallization both theoretically and experimentally. The application of hydrophobic liquids as nucleation promoters or suppressors has rarely been investigated, and provides an underused avenue to explore in protein crystallization. Theoretically, crystal nucleation is regarded as a two-step process, the first step being a local increase in protein concentration due to its adsorption on the hydrophobic surface. Subsequently, the protein is ordered in a crystal lattice. The energetic aspect of crystal nucleation on water/hydrophobic substance interfaces is approached by calculating the balance between the cohesive energy maintaining integrity of the two-dimensional crystal nucleus and the sum of destructive energies tending to tear up the crystal. This is achieved by comparing the number of bonds shared by the units forming the crystal and the number of unshared (dangling) bonds on the crystal surface pointing toward the solution. The same approach is extended to three-dimensional protein crystal nucleation at water/hydrophobic liquid interfaces. Experimentally, we studied protein crystallization over oils and other hydrophobic liquids (paraffin oil, FC-70 Fluorinert fluorinated oil, and three chlorinated hydrocarbons). Crystallizations of α-lactalbumin and lysozyme are compared, and additional information is acquired by studying α-crustacyanin, trypsin, an insulin analogue, and protein Lpg2936. Depending on the protein type, concentration, and the interface aging time, the proteins exhibit different crystallization propensities depending on the hydrophobic liquid used. Some hydrophobic liquids provoke an increase in the effective supersaturation, which translates to enhancement of crystal nucleation at their interface with the crystallization solution, leading to the formation of crystals.

Lysozyme Membranes Promoted by Hydrophobic Substrates for Ultrafast and Precise Organic Solvent Nanofiltration

“…This speculation can be exactly supported by the results of water contact angle measurements (Figure b). Inspired by this process, we suggest that a hydrophobic substrate at the air/water interface would facilitate the heterogeneous nucleation of lysozyme oligomers on its surface and then result in decreased particle size and small pores in the formed nanofilm because of the hydrophobic–hydrophobic interactions. − TEM images display that the oligomers on the surfaces of our LNCM have an average size of ∼15 nm which are smaller than those directly formed at the air/water interface (Figure S8, Supporting Information). Thus, the lysozyme nanofilm shows a geometric pore size of 2.32 nm calculated by a closely packed model.…”

Globular protein assembly and network formation at fluid interfaces: effect of oil

“…[10][11][12][13] Moreover, protein behavior at fluid interfaces gains attention in nutritional, medical, and pharmaceutical research efforts in areas such as protein crystallization, lipid bodies, protein digestion, antibody stability, vaccination efficiency, bio-mimicing protocells, and cell membrane functions. [14][15][16][17][18][19] Protein adsorption is governed by the physicochemical properties of both subphases.…”

Enhancing the crystallisation of insulin using amino acids as soft-templates to control nucleation

“…Recent studies showed that amino acids can impact and alter the secondary structure of the native protein (67) which also indicates a change in the interaction intensity between the protein molecules and bulk liquid phase. Additionally, the promoting effect of hydrophobic additives has already been shown in literature (68)(69)(70). Since leucine is a hydrophobic amino acid and reduces the interactions between the water molecules, the addition of leucine might lead to an increase in the hydrophobic interactions.…”