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Poster Presentations
Day 2, June 23（Mon.）
Room P (Maesato East, Foyer, Ocean Wing)
- 2P-PM-39
Probing Protein Structural Heterogeneity in Living Cells Using In-Cell Mass Spectrometry and Vacuum Ultraviolet Photodissociation
(1DICP, 2UCAS)
oShirui Yang1,2, Zheyi Liu1,2, Fangjun Wang1,2
Conformational ensembles play an important role in protein function. However, the dynamic regulation of these ensembles within living cells remains underexplored with existing technologies. In this study, we develop an in-cell mass spectrometry (MS) combined with ultraviolet photodissociation (UVPD) approach to characterize protein structrual heterogeneity in native cellular environments. Using induced electrospray ionization (iESI), we directly release and ionize intracellular calmodulin (CaM) from E. coli, revealing different conformational distributions influenced by cellular conditions. Ion mobility-MS and UVPD analysis identified compact, extended, and partially unfolded CaM conformation. UVPD fragmentation patterns indicated different structural details and different Ca2+ binding preferences: EF-3/4 in extended CaM and EF-2/3 in compact CaM, highlighting conformation-dependent allosteric regulation. Compared to purified CaM, intracellular CaM exhibited enhanced structural flexibility, emphasizing the role of the native environment in shaping protein conformational landscapes. This investigation demonstrates the importance of in-cell MS and UVPD in resolving structural heterogeneity at the residue level, offering insights into the protein structure–function relationships of flexible proteins in living systems.