article epub_ahead yes Darja Rohden author 81dc668a-19fa-11f0-bf31-d56534059ef3 Federico Napoli author d42e08e7-f4fc-11eb-af0a-d71e26138f1b0000-0002-9043-136X Roman J. Lichtenecker author Paul Schanda author 7B541462-FAF6-11E9-A490-E8DFE56974250000-0002-9350-7606 PaSc department GradSch department AlloSpace. The emergence and mechanisms of allostery project The prevalence of proline residues in intrinsically disordered proteins, functional hinges of enzymes, and protein interaction sites highlights the importance of studying their local dynamics and interactions at atomic resolution. While nuclear magnetic resonance (NMR) spectroscopy is, in principle, ideally suited for such investigations, the absence of an amide proton precludes the use of conventional 1H–15N amide-detected experiments. 1H–13C-based approaches, though possible, remain challenging in large proteins. In this work, we present a selective labeling strategy that introduces an isolated 1H2δ–13Cδ–15N spin system in proline residues in otherwise deuterated proteins. Our method leverages the enzymatic conversion of ornithine, synthesized from cost-effective isotopic precursors. We demonstrate the utility of this approach by applying it to a 134 kDa enzyme using magic-angle spinning NMR, highlighting its potential to efficiently probe functionally relevant sites in proteins. American Chemical Society2026 eng 1554-8929 1554-8937 4286312710.1021/acschembio.6c00587 Rohden D, Napoli F, Lichtenecker RJ, Schanda P. 2026. Chemoenzymatic proline isotope labeling for solution and magic-angle spinning protein nuclear magnetic resonance. ACS Chemical Biology. D. Rohden, F. Napoli, R.J. Lichtenecker, P. Schanda, ACS Chemical Biology (2026). Rohden, Darja, et al. “Chemoenzymatic Proline Isotope Labeling for Solution and Magic-Angle Spinning Protein Nuclear Magnetic Resonance.” <i>ACS Chemical Biology</i>, American Chemical Society, 2026, doi:<a href="https://doi.org/10.1021/acschembio.6c00587">10.1021/acschembio.6c00587</a>. Rohden, Darja, Federico Napoli, Roman J. Lichtenecker, and Paul Schanda. “Chemoenzymatic Proline Isotope Labeling for Solution and Magic-Angle Spinning Protein Nuclear Magnetic Resonance.” <i>ACS Chemical Biology</i>. American Chemical Society, 2026. <a href="https://doi.org/10.1021/acschembio.6c00587">https://doi.org/10.1021/acschembio.6c00587</a>. D. Rohden, F. Napoli, R. J. Lichtenecker, and P. Schanda, “Chemoenzymatic proline isotope labeling for solution and magic-angle spinning protein nuclear magnetic resonance,” <i>ACS Chemical Biology</i>. American Chemical Society, 2026. Rohden, D., Napoli, F., Lichtenecker, R. J., &#38; Schanda, P. (2026). Chemoenzymatic proline isotope labeling for solution and magic-angle spinning protein nuclear magnetic resonance. <i>ACS Chemical Biology</i>. American Chemical Society. <a href="https://doi.org/10.1021/acschembio.6c00587">https://doi.org/10.1021/acschembio.6c00587</a> Rohden D, Napoli F, Lichtenecker RJ, Schanda P. Chemoenzymatic proline isotope labeling for solution and magic-angle spinning protein nuclear magnetic resonance. <i>ACS Chemical Biology</i>. 2026. doi:<a href="https://doi.org/10.1021/acschembio.6c00587">10.1021/acschembio.6c00587</a> 231732026-10-19T11:12:28Z2026-10-19T11:20:33Z