Resolving molecular diffusion and aggregation of antibody proteins with megahertz X-ray free-electron laser pulses

Abstract

X-ray free-electron lasers (XFELs) with megahertz repetition rate can provide novel insights into structural dynamics of biological macromolecule solutions. However, very high dose rates can lead to beam-induced dynamics and structural changes due to radiation damage. Here, we probe the dynamics of dense antibody protein (Ig-PEG) solutions using megahertz X-ray photon correlation spectroscopy (MHz-XPCS) at the European XFEL. By varying the total dose and dose rate, we identify a regime for measuring the motion of proteins in their first coordination shell, quantify XFEL-induced effects such as driven motion, and map out the extent of agglomeration dynamics. The results indicate that for average dose rates below 1.06 kGy μs−1 in a time window up to 10 μs, it is possible to capture the protein dynamics before the onset of beam induced aggregation. We refer to this approach as correlation before aggregation and demonstrate that MHz-XPCS bridges an important spatio-temporal gap in measurement techniques for biological samples.

Real-time swelling-collapse kinetics of nanogels driven by XFEL pulses

“…In particular, it has been recently applied to study the phase behavior of PNIPAm systems (22)(23)(24). The development of x-ray free electron lasers (XFELs) with repetition rates in the megahertz regime enabled the study of sub-microsecond processes (25)(26)(27). This is, for instance, the natural diffusion timescale of nanometer-sized particles dispersed in water.…”

“…Here, we present results from x-ray heating induced collapse and swelling of the PNIPAm shell at different cross-linker concentrations and different shell thicknesses. An increasing number of research focus on real-time dynamics of biological systems at free electron laser (FEL) facilities ( 27 , 28 ). FELs make possible to have a direct and real-time view on phenomena such as protein folding kinetics or phase separation, but, at the same time, pose important challenges in the form of radiation-induced effects and sample damage.…”

In situ aggregation and early states of gelation of gold nanoparticle dispersions

“…31 XPCS is frequently used to study soft matter systems, e.g. , obtaining structural and dynamical information of diffusion and hydrodynamic interactions on microsecond timescales, 32–37 glasses and glass transitions, 38–45 and colloidal gelation. 43,46–53 Recently, we have used XPCS to study the gelation of colloidal gold particles grafted with polyethylene (PEG) based ligands and dispersed in a water–glycerol mixture.…”

Coherent X-ray Scattering Reveals Nanoscale Fluctuations in Hydrated Proteins

“…X-ray photon correlation spectroscopy (XPCS) is a technique that utilizes coherent X-rays and can resolve collective nanoscale dynamics, ranging from microseconds to hours. , XPCS has been demonstrated for a broad range of soft condensed matter systems, including amorphous water where a liquid–liquid transition was observed in the ultraviscous regime . However, due to experimental difficulties in working with radiation-sensitive samples, XPCS of protein systems became possible only recently with optimized experimental procedures. − Previous XPCS studies of lysozyme–water solutions explore the dynamics at room temperature of a pressure-induced liquid–liquid phase separation (LLPS) and viscoelastic coarsening process during gel formation, indicating that simple globular proteins such as lysozyme are capable of forming soft nanostructured protein gels …”