- Monte Carlo Simulation Methods for Studying the Thermodynamics of Ligand Binding & Transfer Processes in Biomolecules (2012)
- The binding and transfer of ligands is of central importance for the function of many biomolecular systems. The main topic of this thesis is the development and application of Monte Carlo (MC) simulation methods for studying complex ligand binding equilibria which can also involve conformational changes. The simulated systems were described by microstates within a continuum electrostatics/molecular mechanics (CE/MM) model of the receptor-ligand system. The CE/MM modeling methodology was improved. The improvements led to more detailed molecular models that enable a more realistic reproduction of system properties and environmental conditions. The developed simulation methods were applied to biomolecular systems whose function involves aspects that are important for the understanding of bioenergetic energy transduction. The results of this thesis are presented in five articles that are published in peer reviewed scientific journals. Manuscript A presents the Monte Carlo simulation software GMCT which was largely developed in this thesis. The software offers a variety of different simulation methods that allow the user to harness the full potential of CE/MM models in the simulation of complex receptor systems. Manuscript B presents a novel theoretical framework for free energy calculations with the free energy perturbation method. The novel framework is more broadly applicable and can lead to more efficient simulations than previous formulations. The derivation of the formalism also led to interesting insights into general statistical mechanics. The formalism was implemented in GMCT and could already be used fruitfully for the free energy calculations presented in Manuscripts C and D. Manuscript C demonstrates the application of free energy measures of cooperativity to study the coupling of protonation, reduction and conformational change in azurin from Pseudomonas aeruginosa (PaAz). Such a coupling is prototypic for bioenergetic systems because it forms the thermodynamic basis of their energy transducing function. PaAz is an experimentally well characterized, small electron transport protein. For this reason, PaAz was used here as model system to demonstrate the usefulness of cooperativity free energies in detecting and quantifying thermodynamic coupling between events in complex biomolecular systems. The results of this study led to new insight that could help to determine the still enigmatic physiological role of PaAz. In Manuscript D, free energy calculations were applied to study the thermodynamics of transport through the ammonium transporter Amt-1 from Archaeoglobus fulgidus (AfAmt-1). Ammonium is the most directly utilizable nitrogen source for plants and microorganisms. AfAmt-1 and its homologues facilitate the transport of ammonia/ammonium across biological membranes in living beings from all domains of life. It is intensely debated how these proteins perform their function and whether ammonia or its protonated form ammonium is actually transported. The study extended upon previous theoretical studies by including the effects of substrate concentration, electrochemical transmembrane gradients, proton-coupled binding equilibria and competitive binding of different ligand species. It was found that the transported species is most likely the ammonium ion. An ammonia/proton symport mechanism that involves a pair of coplanar histidine residues at the center of the transmembrane pore as transient proton acceptor is made plausible by the high genetic conservation of these residues. Manuscript E presents a first application of the microstate description within a CE/MM model to the simulation of the non-equilibrium dynamics of a molecular system. We simulated the re-reduction kinetics of the primary electron donor in the photocycle of the bacterial photosynthetic reaction center from Blastochloris viridis. The simulation results are in very good agreement with experimentally measured data.