Study The Noncovalent Complexes Of Protein By Mass Spectrometry

Biomolecular interactions and recognition is the basis of many biologicalprocesses and phenomena of life. In the process of the durg mechanism, most of thedrugs play a pharmacological role through the interaction with targeting biologicalmacromolecules. Protein is usually the main target of drug in the body, studyproteins and small drug molecules the process and mechanism of the interactionbetween not only helps to understand the mechanism of drugs at the molecular level,but also open a new method for the screening of drugs for specific targeting proteinspilot, development of new drugs and design. Electrospray ionization massspectrometry (ESI-MS) has become the most useful tool in the study of noncovalentprotein complexes.This thesis work focus on the development ESI-MS method forthe study of the interaction of proteins and drug molecules and of proteinconformational changes.In this study we have investigated the interaction between saikosaponins andcytochrome c (Cyt c) by electrospray ionization mass spectrometry (ESI-MS).Saikosaponins are glycosides that consist of saccharides and the sapogenins oftriterpenoids. Seven model molecules of saccharides and triterpenes, namely maltose(Mal II), maltotriose (Mal III), raffinose (Raf), stachyose (Sta), glycyrrhetinic acid(Gly), ursolic acid (Urs) and oleanic acid (Ole) were chosen to perform a series ofESI-MS control experiments for the exploration of the interaction groups insaikosaponins with Cyt c. And then we study the interaction between flavone/isoflavone and Cyt c by electrospray ionization mass spectrometry and moleculardocking. Six model moleculs namely tectoridin(Tec)，hesperidin (Hes)，daizin(Din),daizein(Dein),7-methoxy-4’-hydroxyisoflavone(4H7M),7-hydroxy-4′methoxyisoflavones(4M7H) and4’,7-dimethoxyisoflavone(4M7M) were chosen to demonstrateH-bond is the main intercat force between Cyt c and isflavones/flavones, meanwhilepredict the binding sites on Cyt c. Also we study the interaction betweenmaltooligosaccharides/isomaltooligosaccharides and Cyt c by electrosprayionization mass spectrometry. Twelve model molecules namely maltose (Mal II),maltotriose (Mal III), malto-tetraose (Mal IV), maltopentaose (Mal V), maltohexa-ose (Mal VI), maltohepose (Mal VII), isomaltose(Isomal II), isomaltotriose (IsomalIII), isomaltotetraose (Isomal IV), isomaltopentaose(Isomal V), isomaltohexaose(Isomal VI) and isomaltohepose (Isomal VII) were selected to study the influence of ligand size and structure on the stability of noncovalent complexes.Firstly, we have observed in the ESI mass spectra the formation of Cyt ccomplexes with saikosaponin a/c, and these saccharides, with1:1and1:2stoichiometry. Our results showed that no complex ions of triterpenes and Cyt c wasdetected in the ESI-MS and similar Kd values were obtained for the Cyt c complexesof saikosaponins and saccharides. This demonstrates that the glycosyl moiety in thesaikosaponins is the effective interaction group with Cyt c. We propose thatsaikosaponins and saccharides interact with Cyt c by H-bond. The binding affinity ofthese six ligands with Cyt c is shown to be in the order Ssa> Ssc> Raf, Mal III> Sta≥Mal II. This ESI-MS methodology presented in this study enables us to indentifythe interaction group of ligands and allows the direct determination of bindingconstants.Secondly, we have observed the1:1or1:2complexes that fomed by Cyt c andflavones/isoflavones that had chosen but4’,7-dimethoxyisoflavone. Via the controlexperiment we demonstrate that theses ligands are specific binding on Cyt c. Thebinding affinity of these six ligands with Cyt c is shown to be in the order Tec>Hes> Din> Dein>4H7M>4M7H which is agree with the order of H-bond energythat shown in molecular docking results. So we affirm that flavones/isoflavonesinteract with Cyt c by H-bond. The molecular docking results also shown that thebinding sites on Cyt c are Arg-38Trp-59Gly-41Tyr-48(Tec); His-18Trp-59Lys-79Cys-17(Hes); Arg-38Asn-52Gly-41(Din); Arg-38Asn-52Lys-39(Dein); Gln-16His-18Lys-13(4H7M); His-18(4M7H). This methodology that ESI-MS combinedwith molecular docking presented in this study enables us to investigate theinteractions mode between proteins and ligands，and to predict the binding sites onthe protein.Finally, we have observed in the ESI mass spectra the formation of Cyt ccomplexes with maltooligosaccharides and isomaltooligosaccharides, with1:1and1:2stoichiometry. The stability of the noncovalent complexes is shown in the orderMal III> Mal IV> Mal II> Mal V>Mal VI>Mal VII, Isomal III> Isomal IV> IsomalV> Isomal II> Isomal VI>Mal Isomal VII. And the Cyt c-isomaltooligosaccharidescomplexes are more stable than Cyt c-maltooligosaccharrides. This result haveshown that the stability of noncovalent complexes not only relies on the number offunction group, but also the space structure of the ligands.By using the methodologies presented in this study, we have detected theinteraction group of the ligands, demonstrated H-bond to be the main interaction force between protein and ligands, predicted the binding sites on the protein, andanalyzed the stability of noncovalent complexes affected by ligands size andstructure. These results could guide us to further study on the noncovalentcomplexes formed by protein with ligands, medicine mechanism and so on.