| Autor: |
Lins, Jonas, Miloslavina, Yuliya A., Avrutina, Olga, Theiss, Franziska, Hofmann, Sarah, Kolmar, Harald, Buntkowsky, Gerd |
| Zdroj: |
Journal of the American Chemical Society; 20240101, Issue: Preprints |
| Abstrakt: |
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. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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