Ionic-Liquid-Functionalized Mineral Particles for Protein Crystallization

Abstract

Nucleation is a critical step determining the outcome of the entire crystallization process. Finding an effective nucleant for protein crystallization is of utmost importance for structural biology. The latter relies on good-quality crystals to solve the three-dimensional structures of macromolecules. In this study we show that crystalline barium sulfate (BaSO 4 ) with an etched and/or ionic liquid (IL)-functionalized surface (1) can induce protein nucleation at concentrations well below the concentration needed to promote crystal growth under control conditions, (2) can shorten the nucleation time, (3) can increase the growth rate, and finally (4) may help to improve the protein crystal morphology. These effects were shown for lysozyme, RNase A, trypsin, proteinase K, myoglobin, and hemoglobin. Therefore, the use of BaSO 4 particles enables us to reduce the amount of protein in crystallization trials and increases the chance of obtaining protein crystals of the desired quality. In the context of the underlying mechanism, it is shown that the protein−solid contact formation is governed by the interaction of the polar compartments of the biomacromolecule with the support. The tendency of a protein to concentrate near the solid surface is enhanced by both the hydrophobicity of the protein and that of the surface (tuned by the functionalizing IL). These mechanisms of interaction of biomacromolecules with inorganic hydrophilic solids correspond to the principles of amphiphilic IL−mineral interactions. ■ INTRODUCTIONAnalyzing protein crystals is a way to determine the threedimensional structure of the protein, which provides crucial information regarding its biological function. However, protein crystallization is still a major challenge in modern proteomics. Therefore, new and effective ways of creating good-quality protein crystals are continuously sought, and studies of the underlying mechanisms are highly desired.Heterogeneous nucleation (at some pre-existing surface) can be induced under supersaturation conditions lower than those needed for the onset of homogeneous nucleation (in the bulk of the solution). The former offers kinetic advantages and often results in crystals that are bigger and better-ordered, the characteristics that are necessary for high-resolution protein structure determination. Thus, finding an effective heterogeneous nucleant for proteins is a very promising approach, and both empirical attempts and the respective theoretical studies are constantly ongoing. 1 Works in this field have identified the features of the most successful solid nucleating agents as surface nanotopography (preferentially deep, narrow pores of wide size distribution) and/or its amorphous state. 1−6 Very recently, nonporous ordered surfaces have also been found to be prone to induce crystal growth in model systems where the crystal contacts are governed by short-range interactions (as for proteins). 7 The requirement for those surfaces to act as nucleants is the presence of surface strain. Otherwise, crystalline solids ...

Effect of Infrared Light on Protein Behavior in Contact with Solid Surface

“…Those maxima can be attributed to the structural changes and the release of hydration water upon contact of protein with solid surface [60]. The depths of those maxima are enhanced by IR irradiation, which supports the observation on the effect of IR on protein hydration [23] and the results reported in Fig. 3.…”

“…3 For Lys an increase of sediment volume was observed regardless of the environment. As the IR-induced enhancement of protein hydration has been evidenced previously by FTIR spectroscopy for irradiated lysozyme, myoglobin and haemoglobin in water [23], that increase can be attributed to the swelling of particle-protein network due to enhanced water affinity. For BSA the sediment volume increases in water and NaCl solution, due to enhanced hydration as mentioned above, whereas its large decrease (c.a.…”

“…including protein hydration shell, and on protein-protein interactions. [23] In our previous work [23] we have been monitoring possible LED-induced temperature changes for the same time of exposure for 10 µl water drops. Temperature changes were negligible thus excluding bulk thermal effects of IR.…”

“…Recent findings showed that infrared (IR) radiation can promote structuring of water molecules (effective hydration shell expansion) near hydrophilic interfaces [22] including protein interfacial water [23]. This IR-enhanced hydration was shown to be able to induce attractive interactions between like-charged colloids and biomacromolecules in solution [23,24]. These phenomena were explained by considering water-mediated charge separation (polarization/dipole formation).…”

Protein Crystallization in Ionic-Liquid Hydrogel Composite Membranes

“…Therefore, ILs can be considered as a sea of charges that do not organize into a regular structure, while still maintaining strong associations with each other. ILs have been extensively used as additives in protein crystallization [1][2][3][4][5][6] and many screenings using them are already on the market, i.e., the Ionic Liquid Screen, HR2-214 of Hampton Research.…”