Investigation of Weak Interactions of a Photoenzyme With Triplet Quenchers by Native Mass Spectrometry.

Weak and transient molecular interactions can play crucial roles in various biological and chemical processes but are not easy to study with classical structural methods. Herein, we explore the weak interactions between an artificial triplet photoenzyme, RamR, and two triplet quenchers using native mass spectrometry (nMS). The quenchers significantly affect the enzyme reaction, yet the molecular mechanism remained elusive. We systematically analyzed the protein-quencher complexes in both positive and negative ion modes, revealing small charge state shifts indicative of molecular interactions. Collision-induced dissociation (CID) and surface-induced dissociation (SID) were employed to investigate the binding strength, revealing weak binding without well-defined stoichiometry. Interestingly, the addition of both quenchers resulted in shaper peaks in nMS, suggesting competitive binding with residual buffer contents in solution. Combined with previous functional assays, our result agrees with the hypothesis that the quenchers interact with hydrophobic regions of RamR, potentially altering the protein's surface charge and activity. The findings showed that subtle spectral features in nMS can offer clues for investigating protein-ligand binding involving weak or transient interactions.