Fig. 4.

Solution NMR as a tool to investigate weak, transient protein–protein interactions in Fe–S protein maturation pathways. Weak and transient protein–protein interactions are detected by following backbone NH chemical shift changes occurring in a standard ¹H–¹⁵N HSQC NMR experiment and in a ¹H–¹⁵N IR-HSQC-AP NMR experiment, upon titrating ¹⁵N-labeled protein with the unlabeled protein partner and vice versa. Standard ¹H–¹⁵N HSQC experiments allow the identification of protein–protein interacting regions far from the paramagnetic Fe–S cluster (showed in cyano), and to estimate the dissociation constant (K_d) of the observed interaction. ¹H–¹⁵N IR-HSQC-AP NMR experiment allows to identify protein–protein interacting regions close to the paramagnetic Fe–S cluster (showed in yellow). 1D ¹H NMR experiment provides information on the kind of Fe–S cluster(s) bound or assembled on a target protein or protein–protein complex, on the redox state(s) of the cluster(s), and on the cluster ligands