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 complex of the intracellular environment and maybe influenced by the purification process or buffer conditions.
In a study published in the Journal of the American Chemical Society, a research team led by Prof. WANG Fangjun from the Dalian Institute of Chemical Physics (DICP) of the Chinese Academy of Sciences (CAS), in collaboration with Prof. HUANG Guangming from the University of Science and Technology of China, developed a new method for in-cell characterization of proteins using vaccumultraviolet photodissociationtop-down mass spectrometry (UVPD-TDMS). This method provided an innovative technology for analyzing the heterogeneity of intracellular protein in situ with mass spectrometry (MS).
The team combined in-cell MS with 193-nm UVPD to directly analyze protein structures within cells. This method first employed induced electrospray ionization, which ionizes intracellular proteins with minimal structural perturbation. The charge state distributions of intracellular proteins were then analyzed to determine their conformational ensembles. UVPD was subsequently 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.
The team applied this new method to directly ionize and detect highly expressed calmodulin (CaM) from E.coli cells using induced electrospray ionization (iESI). 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, the team 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+ was regulated by conformation-dependent, with the compact conformation showing a higher affinity for Ca2+ than the extended form. Additionally, the team 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," said Prof. WANG. "By precisely selecting the mass and charge distribution, in-cell UVPD-TDMS enables detailed characterization of intracellular protein variants and conformation, and this method has been successfully demonstrated in the analysis of protein heterogeneity," Prof. WANG added.