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- Title
Not All Binding Sites Are Equal: Site Determination and Folding State Analysis of Gas‐Phase Protein‐Metallodrug Adducts.
- Authors
Eade, Liam; Sullivan, Matthew P.; Allison, Timothy M.; Goldstone, David C.; Hartinger, Christian G.
- Abstract
Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites is challenging. Using intact mass spectrometry and proteomics, we investigated the binding of the antimetastatic agent RAPTA‐C to the model proteins ubiquitin, cytochrome c, lysozyme, and myoglobin. Binding to cytochrome c and lysozyme was negligible. However, ubiquitin bound up to three Ru moieties, two of which were localized at Met1 and His68 as [Ru(cym)], and [Ru(cym)] or [Ru(cym)(PTA)] adducts, respectively. Myoglobin bound up to four [Ru(cym)(PTA)] moieties and five sites were identified at His24, His36, His64, His81/82 and His113. Collision‐induced unfolding (CIU) studies via ion‐mobility mass spectrometry allowed measuring protein folding as a function of collisional activation. CIU of protein–RAPTA‐C adducts showed binding of [Ru(cym)] to Met1 caused a significant compaction of ubiquitin, likely from N‐terminal S−Ru−N chelation, while binding of [Ru(cym)(PTA)] to His residues of ubiquitin or myoglobin induced a smaller effect. Interestingly, the folded state of ubiquitin formed by His functionalization was more stable than Met1 metalation. The data suggests that selective metalation of amino acids at different positions on the protein impacts the conformation and potentially the biological activity of anticancer compounds.
- Subjects
BINDING sites; PROTEOMICS; MYOGLOBIN; PROTEIN conformation; CYTOCHROME c; PROTEIN binding; LYSOZYMES
- Publication
Chemistry - A European Journal, 2024, Vol 30, Issue 28, p1
- ISSN
0947-6539
- Publication type
Article
- DOI
10.1002/chem.202400268