Structural and energetic determinants of function in the heterotrimeric G-proteins
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The heterotrimeric G-proteins are important in signal transduction that triggers important biological functions in cells of living organisms. The use of theoretical models to study these proteins gives additional insight into mechanisms of signal transduction and molecular recognition events in the cell. With that objective in mind, molecular dynamics simulations of a heterotrimeric G-protein reveal structural and energetic differences among the states of its signaling cycle. The simulations are stripped explicit waters and analyzed in search of determinants of function with the help of backbone clustering and continuum electrostatic models such as Poisson-Boltzmann (PB) and generalized-Born (GB). The energies are broken down into contributions from individual components corresponding to the carbonyl and amino part of the backbone as well as the side chain of each residue. The results allow the identification of components that are important for folding and binding. Two interdependent results emerge from this research: continuum electrostatic models facilitate the understanding of biological systems from a theoretical point of view; and the heterotrimeric G-protein in its three states serves as a biologically important model for evaluating electrostatic continuum models. As a preliminary work, an evaluation of the GB continuum model was done by measuring entropic differences in free energy of binding from simulations in continuum and explicit solvent models; this supports the use of explicit solvent for molecular dynamics and GB for measuring electrostatic binding free energies. Explicit water for simulation and continuum electrostatic free energy calculations - with GB and PB - were further tested by simulating a heterotrimeric G-protein in three different states for 100ns of simulation time. The degree to which GB approximates PB was quantified for total binding energies as well as for individual components' contributions to binding energies. GB is corroborated as a reasonably good approximation to PB, but it appears to be most useful as a filter of relevant components to be calculated with the more reliable PB method. The simulations of the heterotrimeric G-proteins were extended to 608ns, and further analysis included clustering and the calculations of folding free energies in addition to binding free energies. Cluster analysis was performed on the backbone of the alpha-subunit revealing structural differences in sub domains and loop regions of the heterotrimeric G-protein in its different states. Differences in binding energies from one state of the heterotrimeric G-protein to the other were used to identify and structurally characterize the interaction of important components. At the end of this work, a detailed description of structural and energetic determinants of function in the heterotrimeric G-protein is given at the atomic level. This information gives important insight into the changes that the heterotrimeric G-protein undergo during signal transduction from theoretical models.