Effects of Chemical Cross-Linking on the Structure of Proteins and Protein Assemblies

Chemical cross-linking is frequently used in structural biology for protein stabilization and probing protein structures, often in combination with mass spectrometry (XL-MS). These applications assume that chemical cross-linking does not significantly perturb the protein structure. Here, we directly tested this assumption by monitoring the time course of small-angle X-ray scattering (SAXS) signals from cross-linked protein samples. We investigated two common cross-linking reagents, bis(sulfosuccinimidyl)suberate (BS3) and formaldehyde (FA), with several protein systems ranging from large microtubule filaments down to globular proteins. Across all the measured protein systems, the results consistently showed that BS3 did not significantly change the protein structures, whereas FA induced rapid and substantial changes, including aggregation. Notably, the impact of FA was dose-dependent, with milder structural effects at the lower concentration of 0.1 wt %. Accompanying XL-MS analyses were consistent with the SAXS observations. Overall, the results recommend that in-solution cross-linking based on NHS ester chemistry should be preferred in structural studies over FA wherever possible. In cases where FA cross-linking is used, lowering the FA concentration is of clear importance, and SAXS could be used to verify the integrity of the structure.