Se-77-C-13 based dipolar correlation experiments to map selenium sites in microcrystalline proteins

Sulfur-containing sites in proteins are of great importance for both protein structure and function, including enzymatic catalysis, signaling pathways, and recognition of ligands and protein partners. Selenium-77 is an NMR active spin-1/2 nucleus that shares many physiochemical properties with sulfur and can be readily introduced into proteins at sulfur sites without significant perturbations to the protein structure. The sulfur-containing amino acid methionine is commonly found at protein-protein or protein-ligand binding sites. Its selenium-containing counterpart, selenomethionine, has a broad chemical shift dispersion useful for NMR-based studies of complex systems. Methods such as (H-1)-Se-77-C-13 double cross polarization or {Se-77}-C-13 REDOR could be valuable to map the local environment around selenium sites in proteins but have not been demonstrated to date. In this work, we explore these dipolar transfer mechanisms for structural characterization of the GB1 V39SeM variant of the model protein GB1 and demonstrate that Se-77-C-13 based correlations can be used to map the local environment around selenium sites in proteins. We have found that the general detection limit is similar to 5 angstrom, but longer range distances up to similar to 7 angstrom can be observed as well. This study establishes a framework for the future characterization of selenium sites at protein-protein or protein-ligand binding interfaces.

Automated summary

Sulfur-containing sites in proteins play crucial roles in protein structure and function. Selenium-77 can replace sulfur and be introduced into proteins without significant structural changes. However, there is currently no established method to accurately map the local environment around selenium sites. This study explores a method using correlations between selenium and carbon-13 to characterize the local environment of selenium sites in a model protein variant.