A multiscale in situ time-resolved study of the nano- to millisecond structural dynamics during protein crystallization.

Protein crystallization is key to determining the structure of proteins at atomic resolution. It can occur naturally, including in pathological pathways, for instance with aquaporin and γ-crystallin proteins. A fundamental understanding of the underlying crystallization process is both technologically and biologically relevant. A multitechnique approach is employed here to investigate protein crystallization in situ, allowing us to assess the evolution of the liquid suspension and crystallite structure as well as protein diffusion during the crystallization process. The wide range of methods probe the sample on ångström to millimetre length scales, accessing nanosecond to millisecond dynamics information while acquiring data with minute-timescale kinetic resolution during crystallization. This process takes several hours from an initial state of monomers or small clusters until the presence of large crystallites. Employing neutron spectroscopy allows us to distinguish different crystallization pathways and to reveal the presence of coexisting clusters during the entire crystallization process. We demonstrate the multitechnique approach on human serum albumin (HSA) proteins crystallized from aqueous solution in the presence of LaCl(3). For this system, the crystallization kinetics can be consistently described by a sigmoid function across all methods, and the kinetics can be controlled by the salt concentration. Moreover, we compare the HSA-LaCl(3) model system with the crystallization behavior of β-lactoglobulin-CdCl(2), which includes a metastable intermediate state.