Exploring The Cation-? Interaction In Siderocalin By Single-molecular Force Spectroscopy

The cation-? interaction is the electrostatic interaction between the positive charge of cation and the electron-rich ? system of arenes.It is a very important non-covalent interaction,and plays important roles in many areas,including protein folding,protein stabilization and molecular recognition and so on.The neutrophil gelatinase-associated lipocalin(NGAL)is a member of the lipocalin family,which has conservative secondary structure and function.It can form a siderocalin-siderophore-iron complex,which is apparently different from other lipocalins.Hence,it's also named as siderocalin.However,previous studies were mainly focused on the physiological significance of NGAL.The molecular stability of NGAL and the response of the cation-? interaction to the force direction are far beyond understanding.Recently,single-molecular force spectroscopy based atomic force microscopy has been increasingly used in molecular interaction researches.It provides high atomic spatial resolution and similar operational condition with the physical environment(i.e.,in the solution).As a result,single-molecular force spectroscopy based atomic force microscopy has became one of the most powerful tools to study intermolecular interaction.We investaged the cation-? interaction's strength with the parallel direction using single molecular force spectroscopy in chapter two,.Two novel fusion proteins,namely cys-GB1-NGAL(Q117C)and(GB1)2-NGAL(P102C),were constructed by mutating the sites on the loop inside the disulfide via site-directed mutagenesis and molecular biology approaches.Then,we built NGAL-catechol-Fe(?)complex in vitro and perform single molecular force spectroscopy experiments for the following two groups of each mutant protein including the control proteins without Fe(?)L3 and the experimental protein with Fe(?)L3.However,little significant change was observed due to the weak cation-? interaction when the force was acted on the parallel direction.In chapter three,we further study the strength of the cation-? interaction on the direction of perpendicularity in the NGAL.First,the mutant proteins,cys-GB1?NGAL(K124C/T82C)and(GB1)2-NGAL(F133C/F122C),were constructed using molecular biology methods.Then,these proteins were carried on protein expression and purification,and the NGAL-catechol-Fe(?)complex were further built in vitro.For each mutant protein,we utlized single molecular force spectroscopy to study vertical force acts on the cation-? interaction.We found that the control and complex of the rest of the mutations unfold basically in the same pathway except the T82C mutation,and the unfolding force covers only twenty growth.It maybe caused by the weak cation-7c interaction below the detection limit of atomic force microscope.In the next step,we plan to switch to another protein that contains cation-? interaction and verify the strength of the catione-? interaction by single molecular force spectroscopy.In summary,we systemically investigated the cation-? interaction in NGAL using single molecular force spectroscopy.It will bring us a deep understanding of the non-covalent interaction in protein structure.