Internal Transportation And Signal Transduction In Functional Proteins

The internal transportation tunnels and signal transduction of protein molecules are closely related to their biological functions;for example,the transportation tunnel of small molecules inside the enzyme controls the substrate uptake and product release as well as the binding of inhibitor and agonist.Therefore,it is possible to regulate the reactivity of enzyme by limiting the transportation of small molecules within the protein.On the other hand,the catalytic activity of enzymes can also be regulated exquisitely by covalent modification of key amino acids or non-covalent binding of small molecules,involving the long-distance signal transduction of allosteric signals in proteins.Internal molecular transportation tunnels of laccase and intramolecular signal transduction of Akt kinase are researched in this thesis.Laccase is involved in a variety of biological and industrial processes.Its application is usually limited by the inhibition of chloride,but chloride ions in many cases can not be avoided,prompting one to enhance laccase's tolerance to chloride.These ions need to pass through a tunnel to reach the trinuclear cluster center which buried deep inside the protein.Here we analyzed the laccase structures generated by classical molecular dynamics simulation using CAVER software and obtained five main tunnels.Then the random accelerated molecular dynamics simulation technique was used to unbiasedly simulate the release process of ligands from the active sites and identified the preferred tunnels of specific ligands.Subsequently,we calculated the free energy distribution of chloride along the five tunnels using the adaptive steered molecular dynamics simulation.The results show that chloride ions are most likely to invade the p1 tunnel.Combining free energy calculations and bioinformatics analysis,theoretical mutagenesis of p1 tunnel-lining residues was carried out,and finally four laccase mutants with high salt tolerance were finally obtained.Akt,as a serine/threonine kinase,is a signal transduction enzyme in many important cellular processes and dysregulation of its activity is the cause of many diseases.However,it remains unclear how the kinase domain integrates different signals and the transition from active conformation to inactive conformation.Here we used molecular dynamics simulations to determine the proper protonated state of H194 in Akt kinase,and described the hydrogen-bonding interaction between G-loop and ATP and the coordination mode of magnesium ions in the active state.The simulation trajectories of ATP/ADP binding model and phosphorylated/ unphosphorylated system found that ADP binding and dephosphorylation of T308 led to the rearrangement of multiple functional elements and the inactivation of kinase.The phosphorylated T308 regulated the binding of ATP and substrate peptide.We found a new allosteric regulatory pathway that further refined the Akt allosteric network.Substrate,product and inhibitor have multiple transport pathways within the laccase and have their own preferred tunnels.After long-term biological evolution,acidic and aromatic residues are significantly enriched around the tunnel in the high-tolerant species which are not conducive to chloride transportation.Phosphorylation of Akt synergizes with the binding of ATP and substrate by allosteric network.The research of these two aspects provides a new idea for further studying the structure-function relationships of proteins and drug design.