Studies Of Superoxide Dismutase Conformation And Interaction With Small Molecules Using Mass Spectrometry

Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease charactered by progressive paralysis and motor neuron death,and still cannot be cured.A large number of studies have shown that the mutation,oxidative modification,misfolding and aggregation of copper zinc superoxide dismutase(SOD1)are closely related to the disease.At present,a commonly recognized cause of the disease is a normal SOD1 destabilize,misfolding or dissociated into monomer,eventually forming the toxic intermediates,and leading to the occurrence of ALS diseases.Therefore,it is of great significance to establish a method to study the SOD1 structure and screen effective inhibitors for treating ALS disease.Due to the complexity of protein complex structure analysis and protein-ligand interaction research system,the existing analytical methods have been put forward with higher requirements.Although traditional structure biological methods such as nuclear magnetic resonance spectroscopy(NMR)and X-ray diffraction crystal method(XRD)can perform high-resolution characterization of biomacromolecular structures,they have limitations on the size,purity of the test sample,sample consumption and so on.Therefore,it is impossible to in-depth study the dynamic changes and complex mechanisms in vivo by single traditional structural biology methods.Compared with these methods,mass spectrometry(MS)has advantages of fast,high sensitivity and providing the information of stoichiometric ratio.Especially,integrated with ion mobility(IM),the dynamic process of protein can be captured by electrospray ionization mass spectrometry(ESI-MS),which can make up the blank of traditional biological methods and has been widely applied in structural biology.In this thesis,native electrospray ion mobility mass spectrometry(Native ESI-IM-MS)was used as the main research method.First of all,the structure stability of normal SOD1 and metal-free SOD1(apo-SOD1)were investigated by collision induced dissociation(CID)and collision induced unfolding(CIU).Results showed that in the same collision voltage and the contents of dithiothreitol(DTT)induced protein instability condition,apo-SOD1 is more likely to dissociate and unfold,implying the SOD1 structure is more stable and the metal ions are one of essential factors in maintaining the structural stability of SOD1.For the poor water solubility of some ligands,in order to screen the effective cosolvents to promote the development of new drugs.the effects of five aprotic polar solvents on the charging,dissociation and unfolding of SOD1 and apo-SOD1 were investigated.Base on CID and CIU mass spectrometry methods,the results showed that 1,3-dimethyl-2-imidazolidinone(DMI),dimethyl-sulfoxide(DMSO)and dimethyl-formamide(DMF)decreased the average charge of protein first and then increased with the increase of concentration,while the dimer content of protein increased first and then decreased.By contrast,acetonitrile(ACN)and tetrahydrofuran(THF)had no similar effects.In order to further analyze the effect of aprotic polar solvent on protein structure,we used DMF and apo-SOD1 as research objects to investigate the effects of DMF on protein dissociation and unfolding by reducing the pH,increasing the cone voltage(CV)and trap collision energy(trap CE).The results showed that at the concentration of DMF from 0%to 1%,the processes of dissociation and unfolding of apo-SOD1 dimer were slowed down,and as the concentration further increased to 20%,the protein structure became unstable.Moreover,the average CCS also revealed that the structure of apo-SOD1 underwent great compaction when the DMF concentration range from 0%to 1%,followed by an extension upon increasing the concentration to 20%,and the corresponding charges first decreased and then increased.Therefore,it is speculated that the conformation stability of DMF-induced protein maybe related to the decrease of coulomb repulsion caused by the decrease of the charge of the protein.On the other hand,the formation of solvent adducts was observed,so the"evaporative cooling"effect of the solvent adduct is also beneficial to the stability of the protein conformation.Stabilization of the conformation of SOD1 by 5-fluorouridine(5-Furd)was studied by Native electrospray ionization mass spectrometry coupled with ion mobility and fluorescence method.The results of ion mobility mass spectrometry indicated that 5-Furd could stabilize the SOD1 configuration,and the fluorescence spectra showed that 5-Furd mainly bound to SOD1 with hydrophobic interaction and can inhibit protein aggregation in vitro.Furthermore,the interaction among the three tea polyphenol compounds and SOD1 were investigated by Native ESI-IM-MS.The binding affinities were mainly assessed by ESI-MS,tandem mass spectrometry(MS/MS),competitive experiment and VC50 method.Results indicated that epigallocatechingallate(EGCG)had the strongest binding affinity with SOD1,and ion mobility results showed that EGCG can slow down SOD1 unfolding.Meantime,spectrum experiment and cell experiment results revealed that EGCG can inhibit the aggregation of SOD1 in vitro and reduced the cell damage.We also studied the interaction between the coffee acyl quinic acid compounds and flavanone compounds with apo-SOD1,the structure of the two compounds and structure-activity relationship of interaction were analyzed.For caffeoylquinic acid compounds,the results showed that the binding affinity of dicaffeylquinic acid was higher than that of monocaffeoylquinic acid,followed by quinic acid and caffeic acid,indicating that caffeoyl maybe a reactive group and the number of caffeoyl groups promote the interaction between the protein and ligand.And an alkali metal ion-assisted ion mobility mass spectrometry method was established to distinguish the isomers.The binding affinities of the dicaffeoyl quinic acid isomers with apo-SOD1 were evaluated indirectly.Furthermore,CIU results indicated that dicaffeoylquinic acid can stabilize the dimer structure of the protein.For flavanones,ESI-IM-MS and MS/MS results showed that the binding affinity of liquiritin apioside was the strongest,better than the corresponding monosaccharide and aglycone,indicating that the presence and the number of glycosyl group was beneficial to improve the binding affinity of the ligand and the protein.The results of fluorescence spectroscopy for inhibiting protein aggregation in vitro were consistent with the binding affitinities.The results of the ion mobility also indicated that liquiritin apioside can slow down the unfolding of the protein.Besides,double-bound liquiritin apioside ligands slowed down the degree of unfolding was better than the single-bound liquiritin apioside ligand.The above results indicated that Native ESI-IM-MS is a powerful tool for screening protein conformation stabilizers and aggregation inhibitors.