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.