Alternative Strategies to Promote Biomolecule Nucleation

Nucleation marks the beginning of phase separation in a system driven to produce crystalline material. Therefore, to determine the final characteristics of the product, crystal density, and size, nucleation should be fine-tuned. This is a general behavior, independent of the nature of the substance. In protein crystallization, the primary goal is typically to obtain crystals of the highest quality possible for structure determination and of a size suitable for the technique to be used. Small homogeneous crystals are queried for XFEL and serial crystallography, whereas neutron diffraction requires them to be cubic millimeter-sized, and electron diffraction needs flat micrometer-sized samples. Therefore, understanding how nucleation occurs and controlling it are of paramount importance not only for crystallizing target enzymes but also for producing biomolecule crystals for pharmaceutical and industrial applications. There are numerous possibilities and strategies for inducing nucleation in protein crystallization, based on various characteristics such as chemical influences, modified surfaces, or physical methods, including the use of external fields, e.g., electric, magnetic, ultrasound, or light. In this chapter, we will cover a portion of these possibilities. We will review the influence of several chemical substances, including hydrogels, modified surfaces, nanoparticles, and ionic liquids. New trends, such as the use of the switchable water concept and crystallization under hydrophobic conditions, will also be introduced as future perspectives.