Assessment on site-specific thiol-based paramagnetic labeling strategies of metalloproteins for NMR and EPR applications.

Paramagnetic NMR and EPR spectroscopy are important biophysical methods in structural biology, and site-specific protein labeling is generally required for their application in biological systems. Thiol chemistry is widely used for site-specific paramagnetic labeling due to its high reactivity, selectivity, and efficiency. Given that more than 30% of proteins require metal ions as cofactors, the compatibility of current thiol-based labeling strategies with metalloproteins containing cysteine-coordinated metal centers requires careful evaluation. Here, we evaluated widely used thiol-based paramagnetic labeling strategies with zinc-binding proteins using the baculovirus IAP repeat (BIR) domain of X-linked inhibitor of apoptosis protein (XIAP) as a model system. The XIAP BIR domain coordinates a zinc ion through three cysteine residues and one histidine residue, forming a zinc finger motif. The reactivity of the zinc-coordinated cysteines toward different paramagnetic tags was evaluated by high-resolution mass spectrometry and NMR spectroscopy. Our results show that the commonly used thiol-reactive groups methanethiosulfonate and maleimide are incompatible with site-specific paramagnetic labeling of zinc-finger proteins, because they react with zinc-coordinating cysteines in a stoichiometry-dependent manner. In contrast, the pyridylsulfonyl thiol-reactive group shows no detectable reactivity toward zinc-coordinating cysteines and represents an ideal moiety for site-specific labeling. The metal-chelating moiety in a paramagnetic tag, like strong open-chain chelating ligand, tends to displace zinc ions from the metalloproteins, thereby exposing the coordinated cysteines for modification. Pre-saturation of these metal-chelating tags with zinc ion enables complete, site-specific labeling without perturbing the native zinc finger motif. Overall, we examined the performance of the commonly used thiol-based reactive groups on paramagnetic labeling of metalloproteins, and established a practical protocol for thiol-based site-specific labeling of metalloproteins, particularly zinc-finger proteins.