77Se-13C 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 (1H)-77Se-13C double cross polarization or {77Se}-13C 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 77Se-13C based correlations can be used to map the local environment around selenium sites in proteins. We have found that the general detection limit is ~ 5 Å, but longer range distances up to ~ 7 Å 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.

Analysis of the isomer ratios of polymethylated-DOTA complexes and the implications on protein structural studies.

A rigidified and symmetrical polymethylated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand bearing four SSSS methyl groups in both the tetraaza ring and the acetate arms (SSSS-SSSS-M4DOTMA) was prepared. The isomer ratio of SSSS-SSSS-M4DOTMA complexed with a series of lanthanide ions was carefully investigated using RP-HPLC and NMR. A square antiprismatic (SAP) configuration was exclusively observed for the early lanthanides, while the twisted square antiprismatic (TSAP) geometry was preferred as the lanthanide ion size decreases. The late lanthanides preferentially adopted the TSAP geometry. One of the pendant arms was modified with a pyridyl disulfide group (SSSS-SSSS-M8SPy) for cysteine attachment and displayed a similar isomer trend as the parent compound, Ln-SSSS-SSSS-M4DOTMA. Covalent attachment to the ubiquitin S57C mutant showed resonances whose intensities are in agreement with the isomeric population observed by RP-HPLC. Furthermore, the NOE experiments combined with quantum chemical calculations have unequivocally demonstrated that the SAP of Pr-SSSS-SSSS-M4DOTMA and Pr-SSSS-SSSS-M8SPy, as well as the TSAP of Yb-SSSS-SSSS-M8SPy are more stable than their corresponding isomers.