Study Of Lanmodulin By ¹⁹F NMR And Development Of Novel ¹⁹F Probes

Proteins are the main undertakers of life activity,and the structure of proteins is directly related to their founction.It is very important to obtain the information of proteins conformational change for elucidating the relationship between proteins structure and their function.Nuclear magnetic resonance(NMR)is a powerful tool for obtaining atomic resolution structure information of proteins in vitro and in vivo.19F NMR has been widely applied to study protein conformational change and interactions in vitro and in living cells because fluorinated aromatic amino acids can be easily incorporated into proteins,high sensitivity,simple relaxation mechanism,low background and their chemical shifts are sensitive to local conformational change.In this work,we first investigated the conformation of LanM and its interaction with Ln3+in vitro,E.coli cells and Xenopus laevis oocytes using biosynthetic labeled 19F NMR probe.However,the overexpression level of Mammalian cells is usually low,and the detection of target proteins is difficult to achieve by biosynthetic 19F labeling in cells.We combined electroporation and chemical tagging to observe proteins in Mammalian cells and the interaction of proteins were studied.The main research contents and conclusions are summarized as follows:The 19F NMR Study of Interaction of Lanmodulin with Rare-Earth IonsLanmodulin(LanM),a 12 kDa protein from M.extorquens,was the newly discovered protein that specifically binds lanthanide ions(Ln3+,Ln=La-Lu,Y).LanM is an intrinsically disordered protein in the absence of Ln3+,and it undergoes a large conformational change to a predominantly helical state(～50%)upon binding with four Ln3+.LanM’s high affinity and selectivity for Ln3+make it to be strong potential candidates for biosening,magnetic resonance imaging(MRI),radiopharmaceuticals,and positron emission tomography(PET)imaging.The conformation of LanM and its interaction with Ln3+in vitro and in living cells are the important basis for the development of these potential applications.Nervertheless,there is a lack of relevant studies.Firstly,we have explored the transition process of natural disorder and compact states of LanM and its interaction with different rare earth ions by 1H-15N HSQC and 19F NMR.There was no essential difference in the binding of LanM to different rare earth ions.The conformaonal transition process of lanM upon binding with Ln3+might be a cooperative process of ions.We found that Fe2+,Ni2+,Ca2+,Zn2+,Cu2+ and Mn2+ also induced the conformational changes of LanM.Then,we have explored the conformation of LanM and its interaction with Ln3+ions in E.coli cells and Xenopus laevis oocytes using 19F In-cell NMR.We found that LanM was disordered in E.coli cells in the absence or presence of Y3+/Sm3+.We identified that phosphate group in cells was the major competitor for Ln3+ with LanM.We also directly observed the LanM conformational change in Xenopus laevis oocytes at time resolved manner.LanM ultimately exist in the unfolded form in Xenopus laevis oocytes.Our results implicated that LanM can hardly fold in cells where an abundance of phosphate group exists.When designing or application of biosensors based on LanM conformational change,the strong competition effect of phosphate group in cells has to be considered.Application of Novel 19F NMR Probe in the Detection of Proteins in Mammalian CellsA series of water-soluble trifluoromethyl tags(containing one or two trifluoromethyl groups)with high reaction efficiency and sensitive chemical environment were developed to improve the detection limit of proteins,in order to promote the general applicability of 19F NMR in mammalian cells.The 19F NMR signal intensity of wPSP-6F-tagged proteins were significantly enhanced for more than 6 times compared to that of biosynthetic labelling of 3-fluorotyrosine.The wPSP-6F labeling is sensitive to chemical environment and can effectively distinguish among different conformations of proteins.Combined with protein electroporation and chemical tagging,the 19F signals of GB1(6.5 Kd),UBQ(7 Kd),CaM(17 Kd)and MBP(40.5 Kd)were successfully observed in HeLa cells.The tagging of wPSP-6F can significantly reduce the detection concentration of proteins to 1μM.In addition,wPSP-6F-tagged GB1-A34F homodimeric protein were observed in A2780 in both monomeric and dimeric forms.Compared to in vitro data,the fraction of GB1-A34F dimerization in cells increased significantly,suggesting that the crowded and complex cellular environment can facilitate the dimerization.The development and application of this 19F NMR method is expecred to promote the study of the stucture and function of large molecular weight proteins in living cells.