Crystallization still represents the bottleneck in the process of protein structure determination at high resolution, despite high-throughput structural genomics programs requiring optimized crystallization strategies regarding crystal quality, time, success rate, reproducibility, and used protein amount. On the other hand, the development of suitable materials for controlled heterogeneous nucleation might facilitate biomacromolecular crystallization in a variety of experimental conditions which are not conventionally fruitful. Here, the possibility to fabricate hydrogel membranes displaying controlled chemical composition and nanostructure and to use them as heterogeneous supports for biomacromolecular crystallization is demonstrated. Diverse gel morphologies are obtained by controlling phase separation kinetics during gel layer formation on membrane support. These composite materials are found to increase the effi ciency of the crystallization process so that crystals with enhanced diffraction properties are produced at lower protein concentration than conventional technique, thus affording the possibility to improve current approaches to protein crystallization and to be adapted to specifi c targets.
Ion-exchange membranes were applied in this work to diffuse ions and heavy atoms inside protein crystals in order to gently perform their derivatization. The ion-exchange process rate for three different ions, bromide (Br − ), platinum (Pt + through PtCl 4 2−), and mercury (Hg 2+ ), was evaluated, allowing to determine the concentration of these ions in the crystal solution over time and to evaluate their effect on the crystals. Nafion and Neosepta AXE01, cation and anion exchange-membranes, respectively, were used for transport of cations and anions to hen egg white lysozyme (HEWL) crystals, selected as model protein. X-ray diffraction analysis of the crystals confirmed the attainment of the derivatives and allowed the ab initio building of the bromide derivative model. Derivatization experiments were also conducted by the conventional method, directly soaking the crystals in the heavy atom solution. It was possible to conclude that the controlled diffusion, regulated by the membrane, increases the crystal's stability, avoiding handling procedures (in situ derivatization) and maintaining a safer environment near the crystals without disturbing the vapor diffusion equilibrium.
In this work, the influence of surface topography on protein crystallization over Nafion® is investigated. Two types of Nafion® based membranes were modified by soft lithographic techniques in order to create different topographies at the micro and nano scale and subsequently tested. From the analysis of the induction time, nucleation and crystal growth rate of Trypsin from Bovine Pancreas, all the patterned Nafion® based membranes show an enhanced nucleation and crystal growth. To provide additional insight to the experimental observations, the wettability properties of the prepared samples and the ratio of the Gibbs free energy of heterogeneous nucleation to homogeneous nucleation were evaluated. The crystallization outcome results from the combined effect of both, the structural and chemical properties of the nucleant Nafion® surface.
In recent years, membrane technology has improved the control of protein crystallization and post-crystallization treatment of protein crystals. Many advancements have been achieved regarding solvent evaporation control, heterogeneous nucleation modulation, diffusion of ligands, and the attainment of a protective environment from the combination of membranes with hydrogel materials. Indeed, membranes allow for finer control of the supersaturation rate and nucleation at lower degrees of supersaturation while also enhancing crystallization kinetics, providing greater stability, and decreasing crystal handling during post-crystallization. This comprehensive review addresses the concept of membrane-assisted crystallization with a particular focus on proteins and the impact of the process on the quality of crystal diffraction. Furthermore, it advocates for the benefits of combining membranes with microfabrication technologies and encourages the innovation of new membrane-devices with high-throughput for crystallographic purposes.
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