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

doi:10.1007/s40242-025-5046-4

Chemical Research in Chinese Universities ›› 2025, Vol. 41 ›› Issue (4): 941-947.doi: 10.1007/s40242-025-5046-4

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

LAI Can^1,2,3, LIU Zheyi^1,2,3, LUO Pan^1,2, JIN Zhixiong¹, ZHAO Heng^1,2, WANG Fangjun^1,2,3

1. CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P. R. China;
2. State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P. R. China;
3. University of Chinese Academy of Sciences, Beijing 100049, P. R. China

Received:2025-03-26 Accepted:2025-04-12 Online:2025-08-01 Published:2025-07-24
Supported by:
This work was supported by the National Key R&D Program of China (Nos. 2022YFA1304601, 2022YFC3400502), the National Natural Science Foundation of China (Nos. 22288201, 32088101, 92253304, 22104139), and the Grants from the Dalian Institute of Chemical Physics, Chinese Academy of Sciences (DICP) (Nos. DICP I202242 and DICP I202228) We thank the staff members of the Biological Mass Spectrometry System (https://cstr.cn/31127.02.DCLS.ESBMS) at the Dalian Coherent Light Source (https://cstr.cn/31127.02.DCLS), for providing technical support and assistance in data collection and analysis.

Abstract

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.

Key words: Ultraviolet photodissociation, Native mass spectrometry, Protein complex, Conformation heterogeneity

LAI Can, LIU Zheyi, LUO Pan, JIN Zhixiong, ZHAO Heng, WANG Fangjun. Conformation Heterogeneity of Protein-Ligand Complexes Revealed by Native Mass Spectrometry and Ultraviolet Photodissociation[J]. Chemical Research in Chinese Universities, 2025, 41(4): 941-947.

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

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