Exploring Sulfur Sites in Proteins via Triple-Resonance H-Detected Se NMR.

NMR spectroscopy has been applied to virtually all sites within proteins and biomolecules; however, the observation of sulfur sites remains very challenging. Recent studies have examined Se as a replacement for sulfur and applied Se NMR in both the solution and solid states. As a spin-1/2 nuclide, Se is attractive as a probe of sulfur sites, and it has a very large chemical shift range (due to a large chemical shift anisotropy), which makes it potentially very sensitive to structural and/or binding interactions as well as dynamics. Despite being a spin-1/2 nuclide, there have been rather limited studies of Se, and the ability to use H-indirect detection has been sparse. Some examples exist, but in the absence of a directly bonded, nonexchangeable H, these have been largely limited to smaller molecules. We develop and illustrate approaches using double-labeling of C and Se in proteins that enable more sensitive triple-resonance schemes via multistep coherence transfers and H-detection. These methods require specialized hardware and decoupling schemes, which we developed and will be discussed.