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The scientific discussion about classical and nonclassical nucleation theories has lasted for two decades so far. Recently, multiple nucleation pathways and the occurrence and role of metastable intermediates in crystallization processes have attracted increasing attention, following the discovery of functional phase separation, which is now under investigation in different fields of cellular life sciences, providing interesting and novel aspects for conventional crystallization experiments. In this context, more systematic investigations need to be carried out to extend the current knowledge about nucleation processes. In terms of the data we present, a well-studied model protein, glucose isomerase (GI), was employed first to investigate systematically the early stages of the crystallization process, covering condensing and prenucleation ordering of protein molecules in diverse scenarios, including varying ionic and crowding agent conditions, as well as the application of a pulsed electric field (pEF). The main method used to characterize the early events of nucleation was synchronized polarized and depolarized dynamic light scattering (DLS/DDLS), which is capable of collecting the polarized and depolarized component of scattered light from a sample suspension in parallel, thus monitoring the time-resolved evolution of the condensation and geometrical ordering of proteins at the early stages of nucleation. A diffusion interaction parameter, KD, of GI under varying salt conditions was evaluated to discuss how the proportion of specific and non-specific protein–protein interactions affects the nucleation process. The effect of mesoscopic ordered clusters (MOCs) on protein crystallization was explored further by adding different ratios of MOCs induced by a pEF to fresh GI droplets in solution with different PEG concentrations. To emphasize and complement the data and results obtained with GI, a recombinant pyridoxal 5-phosphate (vitamin B6) synthase (Pdx) complex of Staphylococcus aureus assembled from twelve monomers of Pdx1 and twelve monomers of Pdx2 was employed to validate the ability of the pEF influencing the nucleation of complex macromolecules and the effect of MOCs on adjusting the crystallization pathway. In summary, our data revealed multiple nucleation pathways by tuning the proportion of specific and non-specific protein interactions, or by utilizing a pEF which turned out to be efficient to accelerate the nucleation process. Finally, a novel and reproducible experimental strategy, which can adjust and facilitate a crystallization process by pEF-induced MOCs, was summarized and reported for the first time.

Exploring Nucleation Pathways in Distinct Physicochemical Environments Unveiling Novel Options to Modulate and Optimize Protein Crystallization