Crosslinking/mass spectrometry analysis of Bacillus subtilis RNAP-Delta-HelD complexes

Crosslinking mass spectrometry was applied to investigate protein contacts and dramatic structural rearrangements triggered by HelD binding to RNAP. Four complexes RNAPΔδΔHelD , RNAPΔδΔHelD-δ, RNAPΔδΔHelD-HelD and RNAPΔδΔHelD-δ-HelD were assembled using RNAP purified from B. subtilis ΔhelDΔropE strain (RNAPΔδΔHelD), and recombinant δ and HelD. The four complexes were crosslinked using a heterobifunctional, photoactivatable crosslinker sulfosuccinimidyl 4,4′‐azipentanoate (sulfo-SDA). Crosslinked protein complexes were analyzed by LC-MS/MS, and crosslinked residue pairs were identified in form of crosslinked peptides.