Conformation Heterogeneity of Protein-Ligand Complexes Revealed by Native Mass Spectrometry and Ultraviolet Photodissociation

Abstract

Protein conformation ensembles are directly influenced by functional ligand bindings. Tracking molecular-level fluctuations in protein conformations is critical for advancing targeted drug development and protein engineering strategies. Here, we utilize native mass spectrometry (nMS) integrated with 193-nm ultraviolet photodissociation (UVPD) to probe the heterogeneous conformations of dihydrofolate reductase (DHFR) upon binding functional cofactor and small-molecular inhibitors. The nMS and UVPD techniques allow for simultaneously profiling the inhibitor binding affinity and conformational modulations of DHFR. Our findings demonstrate that the residual fragmentation yield is closely related to the flexibility of local structures. Conformational adjustments induced by either individual or synergistic binding of cofactors and inhibitors unveil novel heterogeneity in DHFR’s secondary structures, particularly α-helices and β-sheet scaffolds within the adenosine-binding domain. This study introduces a promising strategy for characterizing the conformation heterogeneity of targeted protein with diverse ligand modulations.

Keywords

Ultraviolet photodissociation / Native mass spectrometry / Protein complex / Conformation heterogeneity

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Jilin University, The Editorial Department of Chemical Research in Chinese Universities and Springer-Verlag GmbH