Real-Time Control of Nanoscale Protein Assembly for Further Crystallization Using a Solution Circulating Nanoaggregation Control Apparatus

In this study, a solution circulating apparatus was employed to control the aggregation of protein molecules in situ. The association state of protein molecules was controlled by injecting protein solutions, buffers, and solutes into the device while monitoring the heterodispersity of the molecular nanoaggregates by an apparatus of high-performance heterodyne-based dynamic light scattering. Further, a highly ordered association of protein nanoaggregates was induced that lead to the generation and growth of protein crystals suitable for structural analysis by the X-ray protein crystallography. We could also dissociate protein molecules that had already formed aggregates into mono- or bimodal states and reassociate them to form protein crystals, which contribute to minimizing the amount of protein required for crystallization. Our nanotechnological approach allowed us to carefully monitor and control crystal growth with a focus on the association state of the target proteins. The high-performance DLS device monitors the in situ association process of the target proteins allowing a dynamic response to modify the solution properties and leading to a deeper understanding of protein crystallization and achieving rational control of the processes