Combining Solid-state Nuclear Magnetic Resonance And Molecular Dynamics Simulations To Study The Interaction Mechanism Between Membrane Proteins And Molecules

Membrane proteins play key roles in fundamental biological processes such as energy conversion,transport,signal recognition,and transduction.The physiological functions of membrane proteins are dependent on the interactions between membrane proteins and various molecules.Studying membrane proteins or membrane protein-molecule interactions in natural lipid environments is a difficult task due to the complexity of the environment in which membrane proteins are located.Solid-state Nuclear Magnetic Resonance（ss NMR）enables study the interaction of membrane proteins with substrates in their natural environment,and it can obtain high-resolution complex structures of membrane proteins bound to molecules and sites of intermolecular interactions.However,due to the difficulty of preparing high-quality ss NMR samples and the limitation of signal-to-noise ratio and resolution of ss NMR spectra,resulting in ss NMR often has limitations in application.Molecular dynamics（MD）simulations contribute to the elucidation of membrane protein structure and function,and can be used to study membrane protein dynamics and conformational dynamics,not only in conjunction with experiments,but also in conjunction with structural predictions to study systems where experiments cannot be performed.In this thesis,the interaction mechanisms of membrane proteins with three typical molecules（ions,small molecules,proteins）are investigated by using MD simulations,mainly based on ss NMR results.In the membrane protein-ion interactions,Aquaporins（AQPs）and mercury ions were selected as the objects for study.The ss NMR and MD simulations were used to study and reveal the specific molecular mechanisms of mercury ions in the inhibition effect of AQPZ and the activation effect of AQP6.The semi-quantitative distance constraint of Hg²⁺binding to AQPZ cysteine residues was firstly established based on ss NMR results.MD simulations were then used to reveal the specific regulatory mechanisms of the changes in the hydrogen bonding network of the key residues R189/R196 affected by mercury ions in the two water channel proteins.Based on this study we initially developed a methodology for the study of membrane protein-ion interactions.To validate and refine the method,the above established method was further applied to study the changes in the C155 of rat AQP6 protein after binding Hg²⁺ions.The types of residues that form ligands with mercury ions and the effects on the hydrogen bonding network ofα-helical residues were determined by coordination analysis.Eventually,the key residue M160,which affects the water channel,was identified.In the membrane protein-drug interaction system,mechanosensitive channel of large conductance（Msc L）was used to study the transmembrane transport mechanism of Brilliant Green（BG）small molecule by MD simulations and ss NMR methods.Firstly,MD simulations investigated the binding pocket and transport pathway of BG into the Msc L channel,and identified the specific residues bound by BG molecules in the transport process.SSNMR spectroscopy verified these binding sites.This study extends the application of MD simulations and ss NMR methods to membrane protein-drug small molecule interactions.In membrane protein-protein interactions,the SARS-CoV-2 spike-membrane protein was used to study the binding differences between different host ACE2 and stinger proteins by using SARS-CoV-2 spike-membrane protein and host Angiotensin-Converting Enzyme2（ACE2）.The complexes of SARS-CoV-2 Receptor Binding Domain（RBD）and ACE2were constructed by homology modeling and MD simulation.Molecular Mechanics/Generalised Born Surface Area（MM/GBSA）and Potential of mean force（PMF）methods revealed the affinity between SARS-CoV-2 and ACE2 receptors.Energy decomposition identified key residues of the ACE2 receptor in four susceptible species.It was also found that the analysis of the important hydrogen bonding interaction network between ACE2 and RBD could compensate for the lack of resolved structure.This study also further expands the application of MD simulation research methods to membrane protein-protein systems.In summary,the results of the above study show that MD simulations combined with ss NMR systematically elucidate the mechanism of interaction between membrane proteins and ions and small molecules at the molecular level,while also providing a proper investigation of the mechanism of interaction between membrane proteins and proteins in the absence of experimental data.This study provides a reference for other MD simulation studies of more kinds of membrane proteins.