The NMR Analysis On Chemical Modification Of Protein And Biomacromolecule Interactions In Living Cell

The cell is the basic unit of life,and it is the function venue of biomacromolecules.In complex and crowded cellular environments,the hard-core repulsions and chemical interactions will affect the structure and functions of biomacromolecules.In-cell nuclear magnetic resonance(In-cell NMR)can obtain atomic level resolution information of biomacromolecules in living cells,which will help us to understand how the cellular environment affects the structure and function of biomacromolecules.In this work,In-cell NMR technology and 19F atomic probe were used to analyze chemical modification of protein,protein-protein interactions,stability and interactions of G-quadruplex,which were shown as follows:Phosphorylation dependent ?-synuclein degradation monitored by In-cell NMRVarious protein modifications exist in cells,phosphorylation is one of the most common and important modification.However,the direct and real-time monitoring of protein phosphorylation in living cells is still challenging.Herein,we applied in-cell NMR to detect protein phosphorylation and its consequences.Ser-129 phosphorylated?-synuclein,a Parkinson's disease-related protein,was choose as a research model to track its dephosphorylation process in cells.The results showed that ?-synuclein are rapidly dephosphorylated in cells,and phosphorylation accelerates the degradation of?-synuclein.In-cell NMR is useful for the study of protein phosphorylation modification in cells,which monitors the destiny of phosphorylated protein in real time,and provides complementary new ideas for the study of intracellular protein modification.Crowding cellular environment finely tuned protein-protein interactions in living cells learned by in-cell NMRProtein-protein interactions is essential for multiple proteins collaborating to function and completing physiological processes.Previous studies have shown that in the mimicked cellular environment like high concentrations of polymer,proteins or cell lysates,the protein-protein interactions were affected.However,how the complex environment affects protein interactions in living cells is still unknown.Here,the 19F in-cell NMR is applied to the quantitative characterization of protein dimerization interaction in living cells.By comparing the dimerization of A34F GB1(a dimer formed by the GB1 mutant)in dilute buffer and in cells,we found that the dimerization is greatly stabilized,and the dissociation constant reduces by a factor of 6 in the cellular environmen.The strength of protein dimer interactions can be regulated by changing the surface charge of A34F GB1,and the influence of charge-charge interaction is less obvious in cells than that in buffer.Further investigation of the changes of Gibbs free energy in different environments showed that the negative charge-charge repulsion between cytoplasm and protein dimer play an important role in stabilizing the A34F GB1 dimerization in cells.Our results indicated that the cellular environment is important for protein-protein interactions,and cellular complex nonspecific interactions will affect protein-specific interactions in cells.G-quadruplex stability and ligands interaction in living cells learned by in-cell NMRG-quadruplex is an unusual nucleic acid structure,which plays an important role in physiological processes like telomere stability,gene replication,recombination,expression and regulation.G-quadruplex is related to cancer and other diseases.Previous study shown that ions,macromolecular crowding,and interactions will affect the structure of G-quadruplex in vitro,which highlight the importance of the study of G-quadruplexes in living cells.The stablity of human telomeres fragment(MHT24),thrombin-binding aptamer(TBA)and human vascular endothelial growth factor promoter(hVEGFP)were investigated in biological systems,and how the cell types and ionic conditions influence G-quadruplex stability was analyzed.In addition,MHT24 ligands-binding ability with TMPyP4 and 360A are valuated in Xenopus oocytes by 19F in-cell NMR,and we found that the complex cellular environment can change or destroy interaction between G-quadruplex and ligands,which show the importance of studying G-quadruplexes and its interaction with drugs in cells.