Enhancing Sensitivity of Nuclear Magnetic Resonance in Biomolecules: Parahydrogen-Induced Hyperpolarization in Synthetic Disulfide-Rich Miniproteins #Hyperpolarization

Published: Monday, 27 January 2025 - 10:00 -0400

Author: Thorsten Maly

Lins, Jonas, Yuliya A. Miloslavina, Olga Avrutina, Franziska Theiss, Sarah Hofmann, Harald Kolmar, and Gerd Buntkowsky. “Enhancing Sensitivity of Nuclear Magnetic Resonance in Biomolecules: Parahydrogen-Induced Hyperpolarization in Synthetic Disulfide-Rich Miniproteins.” Journal of the American Chemical Society, December 11, 2024, jacs.4c11589.

https://doi.org/10.1021/jacs.4c11589.

Hyperpolarization of small peptides by parahydrogen-induced polarization (PHIP) to increase the sensitivity of nuclear magnetic resonance (NMR) techniques is well established, while its application to larger biopolymers is still a mainly unexplored area. A particular challenge is the presence of folding-essential disulfide bridges. They tend to form metal complexes, thus hampering catalytic hydrogenation, a prerequisite for PHIP. We applied the PHIP technique to enhance NMR signal intensity in cystine-knot miniproteins�highly ordered peptide architectures covalently stabilized by three disulfides. To achieve PHIP, we introduced an L-propargyl tyrosine label at different positions in three synthetic open-chain variants of a natural trypsin inhibitor MCoTI-II. For the folded cystine knot, we observed NMR signal enhancements of up to 499 in methanol, 307 in a D2O−methanol mixture, and 964 for the cysteine-bearing reduced precursor. Trypsin inhibition assays elucidated that introducing a PHIP label into the terminal regions is preferable to alterations within the functional loop to preserve bioactivity. Substitution of the native tyrosine resulted in the highest bioactivity. A drastic reduction in PHIP enhancement was observed in the presence of trypsin due to slower hydrogenation, conditioned by the accessibility of the label within an enzyme− inhibitor complex.