Uncovering hidden structures in protein crystallization with coherent X-rays

The structure of proteins is essential for understanding their function. X-ray diffraction of protein crystals is a powerful tool to obtain their structure and is used both in structural biology and in industrial applications.
However, producing protein crystals can be challenging. Therefore, it is crucial to comprehend the pathways that lead to and control crystal formation. Classical crystallization theory does not fully capture the crystal formation in many systems. Recently, a new mechanism of crystal formation was observed in ferritin solutions, which cannot be adequately described by previous models. In this new mechanism, proteins form first amorphous aggregates, which afterward undergo a structural change via desolvation and become crystalline. The project explores the hypothesized nonclassical mechanism of protein crystallization by measuring the interactions between proteins and their diffusion during the crystallization by means of coherent X-ray scattering.

• Performed analysis to access diffusion of pure ferritin solutions at different concentration which had been measured on nanometer length scale and microsecond time scales at the European Free Electron Laser (EuXFEL).
• Established Ferritin-Polyethylene glycol phase behavior at low concentration.
• Established working acoustic levitation setup
• Performed preliminary X-ray small- and wide-angle scattering (SAXS and WAXS) measurement of ferritin solutions in capillaries at MAXIV.
• Performed X-ray small- and wide-angle scattering experiments of ferritin crystallization in acoustically levitated droplets at MAXIV.
• Analyzed experimental data from the experimental campaigns at MAX IV.
• Setting up of optical microscope for time resolved measurement of ferritin-PEG drying droplet.
• Microscopy measurement of ferritin-PEG drying droplet
• Prepared experimental campaign at European XFEL to measure protein diffusion in levitated droplets.

The project led to the following results:

In protein diffusion:
o Used Megahertz X-ray Photon Correlation Spectroscopy at EuXFEL to measure ferritin collective diffusion at nanometer length scales and microsecond delay times, approaching the protein interaction time.
o Observed anomalous diffusion at elevated concentrations
o Proved signatures of a crossover between short-time and long-time diffusion regimes, consistent with transient cage effects arising from intermolecular constraints in highly crowded environments.

In protein crystallization:
o We proved that ferritin crystallizes in the presence of polyethylene glycol (PEG) at a wide range of molecular weights.
o A state diagram of the PEG-ferritin system has been established.
o Melting of protein crystals has been seen for large PEG molecular weight (>1000 g/mol)
o Two different mechanism of ferritin crystallization have been observed:
• For PEG 3300 and larger: consistent with classical nucleation theory or two step mechanisms
• For PEG 1000 consistent with slow ordering or two step mechanism

The work performed at European XFEL opens the possibility to measure other biological solutions in crowded state at protein molecular length scales (in the range of nanometers) and microseconds time scales. This fills the experimental gap that was present when accessing the collective diffusion in the microsecond region. This has an impact for crowded protein solutions, with possible applications in molecular biology, materials science, food and pharmaceutical industries.

In the context of crystallization, the knowledge gained with using the acoustic levitator, the ferritin-PEG system, and measuring diffusion of protein with X-ray Free Electron Lasers is going to be combine to perform an experiment in which the diffusion of the proteins in monitored during crystallization in the levitated droplets.

Periodic Reporting for period 1 - CRYSTAL-X (Uncovering hidden structures in protein crystallization with coherent X-rays)

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