Work

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

Year: 2024

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 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 D 2 O–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.

Source: Journal of the American Chemical Society

DOI: 10.1021/jacs.4c11589