Study On The Allosteric Motions And Identification Of Key Residues With Elastic Network Model

With the completion of the human gene sequencing project, the post-genomic time is coming, in which the functional genomics and proteomics are regarded as main research subjects of life science. Protein allosteric motions play important roles in metabolism, signal transduction, gene transcription and enzymatic catalysis of living organism. The long-range allosteric motions are usually mediated by the key residues. Many experimental and theoretic methods have been used to investigate the mechanism of protein allostery. It is difficult for the experimental methods to reveal the whole allosteric process at micro-level. The allosteric process of proteins can be simulated by theoretical method at the atomic level. However, the process of protein allostery often requires across high barriers and the simulation is very time-consuming. Some new sampling approaches should be combined with the simulation method to improve the sampling of protein conformational space, such as amplified collective sampling technique, steered molecular dynamics, etc. To be simplified, a variety of different levels of coarse-grained models have been proposed to study the process of proteins allostery. The paper mainly focuses on the identification of allosteric motion and allosteric site with the elastic network model.Mycobacterium tuberculosis L-alanine dehydrogenase(L-Mt Ala DH) has become a target for anti-TB drug research, and the investigation of the functional movements and the identification of key residues in this protein will help us to understand the mechanism of its functions. The study of ligand-gated ion channels contributes to the treatment of Alzheimer’s disease, Parkinson’s disease and other ion channel disease. To study the relationships between protein structure and function of these two systems is of great biological significance. The main content of this dissertation includes the following two parts:Firstly, the molecular dynamics(MD) method and some related software are introduced. Because the MD simulation methods need to consume a large amount of computing resources, it is urgent to develop simple and effective theoretic methods to study protein allosteric motions. Normal mode analysis(NMA) is a simplified method, which is using mode analysis method of proteins of harmonic vibration analysis, and it greatly simplifies the calculation. Another simplified model, elastic network model(ENM) is a coarse-grained model. And it can avoid the disadvantages of the time consuming of NMA, and can be effectively used to calculate the slow motions encoded in proteins. At the same time, this paper introduces the application of Gaussian network model(GNM) and anisotropic network model(ANM), which are belong to ENM, to identify the functional movements and the key residues of the proteins.Secondly, we studied the domain motions and functionally-key residues of L-Alanine dehydrogenase with the ENM. Mycobacterium tuberculosis L-alanine dehydrogenase(L-Mt Ala DH) plays an important role for the catalysis of L-alanine to pyruvate and ammonia, which has been considered to be a potential target for the treatment of tuberculosis. In the present work, the functional domain motions encoded in the structure of L-Mt Ala DH was investigated by using the GNM and the ANM. The slowest modes for the open-apo and closed-holo structures of the enzyme show that the domain motions have a common hinge axis centered in residues Met133 and Met301. Accompanying the conformational transition, both the NAD-binding domain and the substrate-binding domain move in a highly coupled way. The first three slowest modes of ANM exhibit the open-closed, rotation and twist motions of L-Mt Ala DH, respectively. The calculation of the fast modes reveals the residues responsible for the stability of the protein, and some of them are involved in the interaction with the ligand. Then, the functionally important residues relevant to the binding of the ligand were identified by a perturbation analysis method. Our calculation results are consistent with the experimental observations, which is helpful for our understanding the physical mechanism for the function of L-Mt Ala DH.Finally, study of the functionally crucial residues and allosteric pathway of anesthetic regulation of the Gloeobacter violaceus ligand-gated ion channel by use of ENM. Pentameric ligand-gated ion channels(p LGICs) have been found to be the targets of general anesthetics in the organism; however, the mechanism of anesthetic effects of anesthetics on p LGICs remains elusive. The p LGICs from Gloeobacter violaceus(GLIC) can be inhibited by the anesthetics ketamine. The X-ray crystallography has showed that the ketamine binding site is distant from the channel gate of GLIC. It is still not clear how ketamine control the function of GLIC through the long-range allosteric regulation. In the present work, the functionally key residues and allosteric pathway for the anesthetic regulation of GLIC were identified by using a coarse-grained thermodynamic method developed by our group. In our method, the functionally key sites were identified as the residues thermodynamically coupled with the binding of ketamine. The calculation results are well consistent with the available experimental data. Our study is helpful for the understanding of the mechanism of anesthetic action of ketamine on GLIC through long-range allosteric modulation.