Carbon-13 Centerband-Only Detection of EXchange with Dynamic Nuclear Polarization

The magic-angle spinning NMR technique, Centerband-Only Detection of EXchange (CODEX), can be used to determine the oligomerization state of molecules when combined with site-specific labeling. Calibrated with amino acid crystals, the method is successfully applied to proteins, primarily combined with ¹⁹F labeling. The use of ¹³C spins for CODEX-based oligomer determination in proteins is hampered by limited sensitivity of ¹³C spins due to the low gyromagnetic ratio of ¹³C and the presence of natural abundance background spins which contribute to the observed CODEX decay. The use of CODEX is proposed in conjunction with dynamic nuclear polarization (DNP) at low temperature to increase sensitivity. It is necessary to correct for effects of ¹³C present at natural abundance. A (PDSD) proton driven spin diffusion-based correction is demonstrated to be effective when the isotropic chemical shifts of the natural abundance background are distinct from the labeled site. Using a ¹³C-ζ-phenylalanine-labeled GB1 sample, it is demonstrated that the autocorrelation peak decay observed in a series of PDSD spectra can be utilized to correct for the additional dephasing and recover the expected CODEX decay curve. With ¹³C-γ-phenylalanine labeling and ¹³C-depleted background, mixing times up to 1500 s are demonstrated.