Communication pubs.acs.org/crystal
Real-Time Observation of Protein Dense Liquid Cluster Evolution during Nucleation in Protein Crystallization Robin Schubert,† Arne Meyer,‡ Daniela Baitan,†,‡ Karsten Dierks,‡ Markus Perbandt,† and Christian Betzel*,† †
Institute for Biochemistry and Molecular Biology, The Hamburg Center for Ultrafast Imaging, University of Hamburg, Notkestrasse 85, 22607 Hamburg, Germany ‡ XtalConcepts, Marlowring 19, 22525 Hamburg, Germany S Supporting Information *
ABSTRACT: Controlled navigation in the phase diagram of protein crystallization and probing by advanced Dynamic Light Scattering (DLS) technology provided new information and more insight on the early processes during the nucleation process. The observed hydrodynamic radius distribution pattern clearly reveals a two-step mechanism of nucleation and the occurrence of liquid dense protein clusters, which were verified by transmission electron microscopy. The growth kinetics of these protein clusters, forming distinct radii fractions, is analyzed in real time. Further, the data confirmed that critical nuclei show a distinctly different radius distribution than the liquid dense clusters. The data and results provide experimental evidence that during nucleation, a formation of distinct liquid clusters with high protein concentration occur prior to a transition to crystal nuclei by increasing the internal structural order of these clusters, subsequently.
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higher concentration occurs due to the formation of a dense liquid phase, and second, a transition to higher internal order of these clusters takes place.5,17−20 This two-step nucleation model has additional plausibility, because it allows one to explain the large discrepancy between the predicted nucleation rates by the classical nucleation theory and the experimentally obtained nucleation rates.16,17 Due to the small size of the clusters and low frequency in occurrence in the supersaturated solution, experimental evidence for the hypothesis that the second step during nucleation, a transition to a higher order, occurs inside these clusters is still rare.21−23 This is rooted in the fact that following the nucleation process and early crystal growth by optical microscopy is not feasible, because of the optical resolution limit. Therefore, alternative methods need to be applied to study the nucleation process in detail. For several reasons, dynamic light scattering (DLS) is depicted to be one of the most suitable methods. The size of the particles that can be investigated by DLS covers a large range from one nanometer up to a few micrometers. Additionally, DLS is an extremely sensitive method to detect a small number of larger particles in solution. Consequently, the occurrence of larger particle clusters during nucleation can be detected by DLS, even if the frequency of their occurrence (volume fraction) in the crystallization solution is rather low (
