Rational Modification of a Cross-Linker for Improved Flexible Protein Structure Modeling

Chemical cross-linking/mass spectrometry (XL-MS) has emerged as a complementary tool for mapping interaction sites within protein networks as well as gaining moderate-resolution native structural insight with minimal interference. XL-MS technology mostly relies on chemoselective reactions (cross-linking) between protein residues and a linker. DSSO represents a versatile cross-linker for protein structure investigation and in-cell XL-MS. However, our assessment of its shelf life and batch purity revealed decomposition of DSSO in anhydrous solution via a retro-Michael reaction, which may reduce the active ingredient down to below 90%. To mitigate the occurrence of this degradative mechanism, we report the rational design and synthesis of DSSO-carbamate, which contains an inserted nitrogen atom in the DSSO backbone structure. This modification to DSSO yielded remarkably favorable stability against such decomposition, which translated to higher cross-link and monolink recovery when performing XL-MS on monomeric flexible proteins. Recently, XL-MS has been leveraged against AlphaFold2 and other protein structure prediction algorithms for improved prediction of flexible monomeric multiconformational proteins. To this end, we demonstrate that our novel cross-linker, termed DSSO-carbamate, generated more accurate protein structure predictions when combined with AlphaFold2, on account of its increased recovery of cross-links and monolinks, compared to DSSO. As such, DSSO-carbamate represents a useful addition to the XL-MS community, particularly for protein structure prediction.