糖心视频

Proteins in cells are highly flexible and often exist in multiple conformations, each with unique abilities to bind ligands. These conformations are regulated by the organism to control protein function. Currently, most studies on protein structure and activity are conducted using purified proteins in vitro, which cannot fully replicate the complexity of the intracellular environment and may be influenced by the purification process or buffer conditions.

In a study published in the , a team led by Prof. Wang Fangjun from the Dalian Institute of Chemical 糖心视频ics of the Chinese Academy of Sciences (CAS), collaborating with Prof. Huang Guangming from the University of Science and Technology of China of CAS, developed a new method for in-cell characterization of proteins using vacuum ultraviolet photodissociation top-down mass spectrometry (UVPD-TDMS), providing an innovative technology for analyzing the heterogeneity of intracellular protein in situ with MS.

Researchers combined in-cell MS with 193-nm UVPD to directly analyze protein structures within cells. This method employed induced electrospray ionization, which ionizes intracellular proteins with minimal structural perturbation.

The charge state distributions of intracellular proteins were analyzed to determine their conformational ensembles. UVPD was applied to excite and dissociate protein backbones, generating abundant a-, b-, c-, x-, y-, and z-fragment ions, which are rich in protein structure and interaction features.

Moreover, researchers applied this new method to directly ionize and detect highly expressed calmodulin (CaM) from E. coli cells. They discovered that intracellular CaM existed in three main coexisting conformations, with the extended conformation being significantly more abundant than the form found in purified CaM.

Furthermore, researchers employed UVPD-TDMS to study the binding forms and structural characteristics of different Ca²⁺-binding variants of CaM. They found that the ability of CaM to bind Ca²⁺ is regulated by conformation-dependent, with the compact conformation showing a higher affinity for Ca²⁺ than the extended form.

They also revealed that the first two Ca²⁺ ions preferentially bind to EF-2 and EF-3 in the compact conformation, while the extended form favors binding to EF-3 and EF-4 in the C-lobe of the protein.

"Our study introduces a novel concept for in-cell protein characterization. By precisely selecting the mass and charge distribution, in-cell UVPD-TDMS enables detailed characterization of intracellular protein variants and conformation. This method has been demonstrated to have advantages in the analysis of protein heterogeneity," said Prof. Wang.