High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry

We have used intramolecular cross-linking, MS, and sequence threading to ra pidly identify the fold of a model protein, bovine basic fibroblast growth factor (FGF)-2. Its tertiary structure was probed with a lysine-specific cr oss-linking agent, bis(sulfosuccinimidyl) suberate (BS3). Sites of cross-li nking were determined by tryptic peptide mapping by using time-of-flight MS . Eighteen unique intramolecular lysine (Lys-Lys) cross-links were identifi ed. The assignments for eight cross-linked peptides were confirmed by using post source decay MS. The interatomic distance constraints were all consis tent with the tertiary structure of FGF-2. These relatively few constraints , in conjunction with threading, correctly identified FCF-2 as a member of the beta-trefoil fold family. To further demonstrate utility, we used the t op-scoring homolog, IL-1 beta, to build an FCF-2 homology model with a back bone error of 4.8 Angstrom (rms deviation). This method is fast, is general , uses small amounts of material, and is amenable to automation.