Rational Modification\nof a Cross-Linker for Improved\nFlexible Protein Structure Modeling

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