Relaxation-optimized correlation spectroscopy ROCSY for assigning 1H or 13C spin systems in large proteins

Solution NMR studies of large systems are hampered by rapid signal decay. We hereby introduce ROCSY (relaxation-optimized total correlation spectroscopy), which maximizes transfer efficiency across J-coupling-connected spin networks by minimizing the amount of time magnetization spends in the transverse plane. Hard pulses are substituted into the Clean-CITY TOCSY pulse element first developed by Ernst and co-workers, allowing for longer delays in which magnetization is aligned along the z-axis. This has the following consequences: 1) transverse relaxation is minimized, 2) resonance offset effects are minimized, 3) and through-space nuclear Overhauser enhancement (NOE) adds to J-coupling-mediated magnetization transfer. The major drawbacks of the technique are additional heat generation produced by the hard pulses and complication of analysis by the additional through-space NOE magnetization transfer. We demonstrate H_alpha-to-HN correlations not possible using conventional ¹H-TOCSY in a ¹⁵N-enriched sample of PagP (a 161-residue integral membrane protein) in dodecylphosphocholine detergent micelles. We also demonstrate enhanced signal-to-noise compared to ¹³C-TOCSY in a ¹⁵N,¹³C-enriched sample of cardiac troponin C N-terminal domain. We thus propose that ROCSY can be used to boost signal in any protein NMR experiment that utilizes TOCSY, with greater enhancements seen in higher molecular weight systems.