The Preliminary Study On The Interaction Between Methionine Sulfoxide Reductase A And Medicinal Molecule

Background:Methionine sulfoxide reductase A(MsrA),a specific oxidoreductase which is widely distributed in animal and plant cellular,involved in aerobic conditions for cell defense and anti-aging process,is an important intracellular antioxidant barrier.It is involved in the pathological process of many diseases,recently discovered that the pathological process of myocardial ischemia reperfusion,Alzheimer's disease,cataract disease is closely related with MsrA,and so many substrates involved.Atherosclerosis(As)is inflammation-mediated and associated with oxidative stress in chronic cardiovascular disease,which causes cardiovascular disease is now the world's highest morbidity diseases.Many flavonoids have good therapeutic effects on cardiovascular disease,Alzheimer's disease,cataract,and Parkinson's disease.Well-Known proanthocyanidins(PC),quercetin,glycyrrhizin,isoliquiritigenin is widely found in plants of biological flavonoid compounds.Further study of modern pharmacology showed that this kind of bioflavonoid compounds have a wide range of anti-inflammatory and antioxidant Pharmacological activity,such as inhibiting tumor,improveing microcirculation,prevention and treatment of cardiovascular diseases,the protection of brain and nerves,skin care and other functions.Molecular biology researchs has shown that flavonoids play a functional target molecule for the protein,play the essence of activity is the the weak mutual Force between drug and the target protein,and its activity depends on the level of their interaction with the target molecules and the level of the affinity,and its activity depends on the level of their interaction with the target molecules and the size of the affinity,and the level of drug's activity depends on the type of force between molecules and the level of the affinity.Research shows that these four kinds of small organic molecules have anti-inflammatory and antioxidant activity,and MsrA is an important participant in inflammatory and oxidative stress.The recombinant human prokaryotic expression vector pET28a/hMsrA has been successfully constructed in our laboratory.Our research will confirme that the small molecule drugs could interact with MsrA and affect the biological activity of MsrA.Methods:We transformed pET28a/hMsrA recombinant plasmids into E.coli BL21DE3 to obtain the desired hMsrA Strains.Proteins expression were induced by IPTG,high pressure sonication and Ni-NTA affinity chromatography and purification were used to obtain high purity active hMsrA protein.The protein expression level of hMsrA was identified by 10%SDS-PAGE gel electrophoresis.The activity of hMsrA protein was measured with specific substrate Fs.We use fluorescence spectroscopy to analyzes small drug molecules interacting with MsrA in the aspects of the fluorescence quenching spectroscopy,mechanism of fluorescence quenching,apparent binding constants and binding sites,thermodynamics and force analysis.BIAcoreT200 molecule interaction systematically analyzes the affinity kinetic parameters of the intermolecular interactions.Results:(1)SDS-PAGE gel electrophoresis and protease activity test showed that Ni-NTA affinity chromatography could obtain high activity and high purity hMsrA protein.(2)A):Fluorescence spectroscopy titration experiment showed that the fluorescence emission peak of MsrA is regularly reduced along with the flavonoids of proanthocyanidins,quercetin,isoliquiritigenin and glycyrrhizic acid concentration increased in the condition of 280 nm excitation wavelength,indicating that these small molecules can interact with MsrA.However,there was a difference between isoliquiritigenin and other three kinds of small molecules.With the increasing concentration of isoliquiritigenin,the fluorescence peak position of MsrA shifted blue,indicating that the polarity microenvironment of the aromatic amino acid residues of MsrA protein have been changed and formed a complex when isoliquiritigenin interaction with MsrA.B):With the increase of temperature(291K,298K,310K,315K),the of four flavonoid small molecules interaction with MsrA induced quenching constant KQ decrease,indicating that The quenching process of the four flavonoids interaction with MsrA is mainly static quenching.C):Draw the double logarithm of Lg[(FO-F)/F]?Lg[Q]of the small molecule of flavonoids and MsrA at different temperatures,and obtain the corresponding Kb and n according to the intercept and slope.The results show that the binding sites of MsrA interacting with these four flavonoids are close to 1,indicating that MsrA and these four small molecules can form one binding site.The binding kinetics of procyanidins,quercetin,glycyrrhizin,isoliquiritigenin and MsrA is above the order of 104,which indicates that these flavonoids can have a strong binding force with MsrA.D):Thermodynamic analysis found that the free energy change level of the interacting betwween the four classes of flavonoids and MsrA is less than zero,indicating that the combination of these four small molecules and MsrA was an exothermic process and was a spontaneous process.The interaction between isoliquiritigenin,glycyrrhizin,quercetin and MsrA is mainly hydrophobic,and the interaction between procyanidins and MsrA is mainly hydrogen bond and van der Waals force.(3)BIAcore T200 molecule interaction system analysis of four small molecules and methionine sulfoxide reductase A interaction dynamics?affinity.(4)The interaction of the four small molecules drug with methionine sulfoxide reductase A changes the activity of methionine sulfoxide reductase AConclusion:This kind of bioflavonoids with anti-inflammatory and anti-oxidation can interact with methionine reductase A and change the activity of methionine reductase A,which further affects its activity in the body.Our study provides a better biochemical direction for exploring the biological activity of methionine reductase A.