# Carbon‐13 Centerband‐Only Detection of EXchange with Dynamic Nuclear Polarization

## TL;DR

This paper introduces a method to improve the sensitivity of NMR techniques for studying protein oligomerization using 13C labeling and dynamic nuclear polarization.

## Contribution

The novel combination of CODEX with DNP and background correction enables longer mixing times and better sensitivity for 13C-based oligomer analysis.

## Key findings

- DNP and cryogenic conditions significantly enhance 13C-CODEX sensitivity.
- A PDSD-based correction effectively accounts for natural abundance 13C background.
- 13C-depleted labeling further improves the method's performance.

## Abstract

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.

A combination of dynamic nuclear polarization‐based signal enhancement and cryogenic conditions enables counting of spins in a cluster in proteins using 13C‐Centerband‐Only Detection of EXchange when combined with background correction. Optionally, 13C‐depleted labeling of background spins further improves the method.© 2026 WILEY‐VCH GmbH