A ¹³C^β CEST experiment with improved sensitivity for the characterization of protein excited states

Protein function often relies on transient excursions from highly populated ground states to sparse conformers, or 'excited states'. Unlike most other biophysical methods, NMR spectroscopy provides an avenue for quantifying the kinetics and thermodynamics of such transitions and for obtaining structural insights into the participating conformers through measurement of chemical shifts using experiments such as Chemical Exchange Saturation Transfer (CEST). As in many NMR applications, a trade-off between sensitivity and resolution requires compromises that can negatively impact the quality of the resulting data. One example is the ¹³C^β CEST experiment which is often recorded using a triple-resonance scheme with readout of backbone amide correlations. The sensitivity hit associated with multiple magnetization transfer steps challenges applications to protein systems with large linewidth contributions from the exchange process that is the focus of the measurements. Here we present a non-constant time ¹³C^β-¹H^β version of the ¹³C^β CEST experiment that is optimized on a per-residue type basis to provide significant sensitivity improvements over other pulse schemes. The large sensitivity gains in an application to the Q54V variant of pro-interleukin-18 were critical for determination of ¹³C^β chemical shifts for residues interconverting between the ground state and a pair of excited conformers. Technical details of the experiment are presented.