Enhancing Sensitivity\nof Nuclear Magnetic Resonance\nin Biomolecules: Parahydrogen-Induced Hyperpolarization in Synthetic\nDisulfide-Rich Miniproteins

Hyperpolarization of small peptides by parahydrogen-induced\npolarization\n(PHIP) to increase the sensitivity of nuclear magnetic resonance (NMR)\ntechniques is well established, while its application to larger biopolymers\nis still a mainly unexplored area. A particular challenge is the presence\nof folding-essential disulfide bridges. They tend to form metal complexes,\nthus hampering catalytic hydrogenation, a prerequisite for PHIP. We\napplied the PHIP technique to enhance NMR signal intensity in cystine-knot\nminiproteinshighly ordered peptide architectures covalently\nstabilized by three disulfides. To achieve PHIP, we introduced an l-propargyl tyrosine label at different positions in three synthetic\nopen-chain variants of a natural trypsin inhibitor MCoTI-II. For the\nfolded cystine knot, we observed NMR signal enhancements of up to\n499 in methanol, 307 in a D<sub>2</sub>O–methanol mixture,\nand 964 for the cysteine-bearing reduced precursor. Trypsin inhibition\nassays elucidated that introducing a PHIP label into the terminal\nregions is preferable to alterations within the functional loop to\npreserve bioactivity. Substitution of the native tyrosine resulted\nin the highest bioactivity. A drastic reduction in PHIP enhancement\nwas observed in the presence of trypsin due to slower hydrogenation,\nconditioned by the accessibility of the label within an enzyme–inhibitor\ncomplex.