Following protein dynamics in real time during crystallization


Diffraction from protein crystals remains the central pathway to high-quality protein structure determination, but a universal approach to crystallization has not been presented. This lack is linked to the underlying fundamental complexity of protein crystallization: proteins are nonspherical, flexible macromolecules with highly anisotropic interactions.
Anisotropic sticky attraction can induce amorphous aggregation, and a better understanding of the non-equilibrium pathways of crystal nucleation is of utmost importance for a predictive picture.
In this context, information on the molecular dynamics of proteins during the crystallization process is highly desirable.


We employed real-time monitoring of nanoscopic dynamics using neutron spectroscopy and structure using SANS on protein solutions prone to crystallization. At the neutron backscattering spectrometer IN16B (ILL), we collected spectra dominated by the incoherent proton scattering with a spacing of 15 min. These data were complemented with time resolved coherent scattering at the neutron spin echo spectrometer IN11 (ILL), where we focused on the dynamics on and beside the crystal Bragg peak. Combining all techniques, we obtain a consistent picture on the fraction and mobility of proteins both left free in solution and incorporated into crystals.

Figure 1. Figure 1 - Time-resolved spectra of protein dynamics from neutron backscattering spectroscopy. The protein samples crystallized during the time span of the experiment, causing a significant change in the spectra shape (inset left: first spectrum; inset right: last spectrum), which can be used in model fits to quantify dynamics and fractions of proteins in solution and in the crystal.


Besides a more systematic understanding of dynamical effects of e.g. crystallization agents, the established methodology opens more generally opportunities for studies on the temporal evolution of nanoscopic dynamics in biological and soft matter systems.


The work was done in a collaboration between researchers at the ILL, the University of Tübingen, and the University of Lund. All measurements were performed at ILL, France. The study was supported by a binational DFG-ANR grant and the KAW Foundation. The study profited from PhD studentships funded by the ILL and the
University of Tübingen.

Further information:
Chr. Beck, M. Grimaldo, F. Roosen-Runge et al.
Cryst. Growth Des. 2019, 19, 7036−7045
DOI: 10.1021/acs.cgd.9b00858


Felix Roosen - Runge, Malmö University


Tilo Seydel, Institute Laue Langevin