Pure shift amide detection in conventional and TROSY-type experiments of 13C,15N-labeled proteins

Large coupling networks in uniformly C-13,N-15-labeled biomolecules induce broad multiplets that even in flexible proteins are frequently not recognized as such. The reason is that given multiplets typically consist of a large number of individual resonances that result in a single broad line, in which individual components are no longer resolved. We here introduce a real-time pure shift acquisition scheme for the detection of amide protons which is based on C-13-BIRDr,X. As a result the full homo- and heteronuclear coupling network can be suppressed at low power leading to real singlets at substantially improved resolution and uncompromised sensitivity. The method is tested on a small globular and an intrinsically disordered protein (IDP) where the average spectral resolution is increased by a factor of similar to 2 and higher. Equally important, the approach works without saturation of water magnetization for solvent suppression and exchanging amide protons are not affected by saturation transfer.

Automated summary

We present a real-time pure shift acquisition scheme for the detection of amide protons, which can effectively suppress the full homo- and heteronuclear coupling network, leading to improved resolution and sensitivity.