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

TL;DR

This study uses megahertz X-ray photon correlation spectroscopy at XFELs to investigate antibody protein dynamics, identifying optimal dose regimes to avoid radiation-induced aggregation and enabling new insights into biological macromolecule behavior.

Contribution

It demonstrates the feasibility of measuring protein dynamics with MHz-XPCS while avoiding beam-induced aggregation, advancing the understanding of radiation effects in biological samples.

Findings

Protein motion can be measured before aggregation occurs at dose rates below 1.06 kGy/μs.

Identified a regime for capturing antibody protein dynamics in dense solutions.

Demonstrated MHz-XPCS as a tool bridging a spatio-temporal gap in biological measurements.

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 $1.06\,\mathrm{kGy}\mathrm{\mu s}^{-1}$ in a time window up to $10\,\mathrm{\mu s}$, 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.