Structural And Functional Characterization Of Urea Transporter, DvUT, And Chloridion Transporter, EcClC

Channels are common protein in biological system. They are tiny pores that are distributed on the surface of cells. And the main function of channels is facilitating polar small molecules or ions fluxing into or out of cells. For past few years, great progress has been made in defining their molecular identity and basic functional characteristics by experimental studies, and many crystal structures of channels have been solved. It greatly promote the theoretical modeling and simulation studies. In this thesis, based on the crystal structures, we study the structural and functional characterization of two channels, Desulfovibrio vulgaris urea transport, dvUT, and Eschericia coli chloridion transporter, EcClC, with molecular dynamic simulation, Monte Carlo simulation, and modeling and simulation techniques.1. Computation and simulation of the structural characteristics of the urea transporter and behaviors of urea transportUrea transporters are a family of membrane proteins that transport urea molecules across cell membranes and play important roles in a variety of physiological processes. Although the crystal structure of bacterial urea channel dvUT has been solved, there lacks an understanding of the dynamics of urea transport in dvUT. In this study, by using molecular dynamics simulations, Monte Carlo methods, and the adaptive biasing force approach, we built the equilibrium structure of dvUT, calculated the variation in the free energy of urea, and determined the urea-binding sites of dvUT, gained insight into the microscopic process of urea transport and studied the water permeability in dvUT including the analysis of a water chain in the pore. The strategy used in this work can be applied to studying transport behaviors for other membrane proteins.Using MD and MC simulation methods we studied the structural characteristics and transport behaviors of dvUT. By the aid of computed binding free energy and retention probability of urea molecule along the channel we determined the positions of three urea binding sites, which are located at z=-5.0 A,4.0 A, and 9.0 A for the external (Sext), middle (Smid), and internal (Sint) binding sites, respectively. These binding sites are separated by the energy barriers, and the sites participate in multiple hydrogen bonding interactions with the urea molecule. Our MD simulations suggested that dvUT is water permeable, and the average rate is about 0.4 H2O/ns/channel for the water molecules to cross the dvUT pore. The movement of urea is always accompanied by water. We calculated the probability density of the water molecules in the dvUT pore and the projection of the dipole moments of the water molecules along the axis of the pore. We found that the pseudo-two-fold symmetry of the wall of the pore was the reason for the water molecules on the two sides of the midpoint of the pore to have opposite dipole moment orientations.2. Modeling and simulation of the functional characterization of the urea transporterWe constructed a cooperative binding model and established a mutual substitution rule for binding and substituting of urea and its analogue DMU in UTs. Based on built equilibrated structure of dvUT and computed retention probability of urea molecule along the channel, using the cooperative binding model, the substitution rule and MC simulations, as well as modeling and simulation techniques, we reproduced the experiment data points of urea flux through the dvUT, equilibrium binding of urea to dvUT, and the mutual substitution of urea and DMU in dvUT. The modeling uncovered the microprocess of these biochemical experiments. This investigation provides a new way to unify the study of structures and functions of proteins based on modeling and simulation.Furthermore, our simulations suggested that there are only two DMU-binding sites per dvUT, and explained the experimental finding that relatively small amounts of DMU can displace more urea in dvUT. This suggestion is consistent with the crystal structure of dvUT complexed with DMU. The urea-DMU substitution behaviors can be regarded as DMU competing with urea for the binding sites in dvUTs, which is determined by the cooperativity parameter and the retention probability profile of urea in dvUT.3. Calculation and simulation of the behaviour of EcClC dimer separating into two monomers influenced by mutating the interface's residuesThe chloridion transporter, EcClC, is a homodimmer, which consist of two similar monomers. Shape complementarity is the important factor for the monomer closely connecting with the other. Based on the crystal structure of EcClC, we mutate the I or L residue into W residue on the interface of the two monomers. Since the size of W residue side-chain is larger than that of I/L residue side-chain, the mutated residue favors the interface's exposure to the lipid bilayer, and plays the role of two aspects of the dimer separation, 'warts-and-hooks'.We establish five mutant residue-membrane-solution system,1201W, L406W, I422W, L434W,1201W/I422W (WW) system, and makes simulation for-22ns for each system. We calculate the interface area between the two monomers depending on time, which represents the separating tendency of the dimmer. We find that the separating tendency is obvious in I422W and WW systems. In the WW system, the mutant residues 1201W is located in the middle of the interface, and I422W is located on the edge of the interface. Both 1201W and I422W play the role only as 'warts'. However, the main reason for separation in the I422W system is the I422W residue in one monomer turning to membrane, which plays the role as 'warts-and-hooks'.