Study On The Interaction Of Ligands With β-lactoglobulin By Native Electrospray Ionization Mass Spectrometry And Molecular Docking Calculation

Proteins are one of the six major elements of normal life activities in human body.They participate in the renewal and repairment of tissues and the regulation of physiological functions and often achieve their physiological functions through the interaction with other ligand molecules.Non-covalent interactions between proteins and their ligands are also one of the most basic biological processes in organisms.Studying and understanding the interactions between protein and ligands can provide insights into the effects of ligand binding on protein structures and functions,which would be helpful for molecular biological effects and toxicity mechanisms.In addition,protein-ligand interaction research also has important guiding significance for drug discovery,design and development.In this thesis,the interactions ofβ-lactoglobulin and three extracted components of snake grass and two perfluorinated compounds,were analyzed using Native Electrospray ionization-mass spectrometry(ESI-MS)and hydrogen-deuterium exchange mass spectrometry(HDX-MS),respectively.Molecular docking simulation calculations were conducted to study the interaction between aromatic hydrocarbon receptors and polybrominated diphenyl ethers and established a QSAR model that predicted the binding ability of the two at the molecular level.This study of the interactions between proteins and ligands is helpful for drug design and development and elucidates the toxicity mechanism of pollutants.1)Using non-denaturing electrospray mass spectrometry,the interactions betweenβ-lactoglobulin and three kinds of snakeroot extracts were studied.These three types of snakeroot extracts all have a tetrahydro-β-carolinone structure,which is considered to have anti-tumor activity and therefore they may have potential anti-tumor properties.It was observed by ESI-MS that they formed a complex withβ-lactoglobulin at a molar ratio of 3:1.The order of binding ability was measured as:5-oxodolichantoside>ophiorrhiside H>ophiorrhiside G.Competitive experiments on multiple binding sites ofβ-lactoglobulin revealed that 5-oxodolichantoside and ophiorrhiside H mainly bound withβ-lactoglobulin in its central hydrophobic cavity,whereas ophiorrhiside G bound in the central hydrophobic cavity and the hydrophobic region on the surface of the protein.Oxodolichantoside and ophiorrhiside H binding were greatly affected by electrostatic effect,while ophiorrhiside G binding was less affected by electrostatic effect.Molecular docking studies revealed that5-oxodolichantoside andβ-lactoglobulin formed more hydrogen bonds.The results of circular dichroism showed that 5-oxodolichantoside had the greatest influence on protein structure.Studying and understanding the non-covalent interactions betweenβ-lactoglobulin and these three natural extracts is important for the design and development of drugs.2)The interactions betweenβ-lactoglobulin and two perfluorinated compounds were studied by a combination method of hydrogen deuterium exchange and electrospray mass spectrometry.The addition of PFOA and PFOS led to a reduction of 10 deuteriums inβ-lactoglobulin.This indicated that PFOA and PFOS did indeed bind toβ-lactoglobulin,which resulted in a tighter protein conformation.The inflection point of the reduced number of deuterations indicated that the binding dose ratio was 3:1,showing that there may be an exchange of ligands at different sites.Through the kinetics study of the hydrogen-deuterium exchange process,the addition of PFOA and PFOS slightly increased the rate of hydrogen-deuterium exchange,for which the deuterium exchange rate of the protein and PFOS complex was slightly greater than that of PFOA.Molecular docking found that PFOS could interact withβ-lactoglobulin.Combining more hydrogen bonds led to less exchangeable amide hydrogen.PFOA and PFOS are widely present in the environment and have a strong interaction withβ-lactoglobulin.Therefore,β-lactoglobulin may be used as a carrier for perfluorinated compounds and participate in the biological processes in vivo.Study on the interactions ofβ-lactoglobulin and perfluorinated compounds helps to understand the mechanism of toxicity of perfluorinated compounds.3)The structure-activity relationship model of the binding affinity between the polybrominated diphenyl ether molecule and the aromatic hydrocarbon receptor was established through machine learning.The 2D-QSAR model established the relationship between the binding affinity and the binding affinity according to the descriptor.The R~2of the model reached 0.918,which had a good Predictive power.3D-QSAR was modeled according to the COMFA and COMSIA methods.The R~2of the model was above 0.98,which was better than 2D-QSAR’s predictive ability.According to the analysis of electrostatic field,stereo field and hydrophobic field,the substitution of Br atoms at 3’or 4’position was conducive to the increase of binding affinity,while Br at 6’position was not conducive to the increase of binding affinity.Polybrominated diphenyl ethers are a widespread persistent organic pollutant.The toxicity mechanism in organisms is still unclear.Studying the interaction between aromatic hydrocarbon receptors and polybrominated diphenyl ethers helps to understand the toxic mechanism of polybrominated diphenyl ethers and the different structures of polybrominated diphenyl ethers.