MBAR-enhanced lattice Monte Carlo simulation of the effect of helices on membrane protein aggregation

We study the effect of helical structure on the aggregation of proteins using a simplified lattice protein model with an implicit membrane environment. A recently proposed Monte Carlo approach, which exploits the proven statistical optimality of the MBAR estimator in order to improve simulation efficiency, was used. The results show that with both two and four proteins present, the tendency to aggregate is strongly expedited by the presence of amphipathic helix (APH), whereas a transmembrane helix (TMH) slightly disfavours aggregation. When four protein molecules are present, partially aggregated states (dimers and trimers) were more common when the APH was present, compared with the cases where no helices or only the TMH is present

Advances in Monte Carlo techniques with application to lattice protein aggregation

Motivated by an intricate mechanism to transport folded proteins across biological membranes, known as the Twin-arginine translocation (Tat) pathway, we construct lattice protein models in an attempt to study the aggregation of the membrane protein TatA, which plays an integral role during active Tat translocation. We develop force field that characterizes intra- and inter-residue interactions, as well as how each residue interacts with its environment. Although written with the Tat process in mind, this thesis is mainly devoted to developing efficient Monte Carlo schemes for biomolecular simulations, which are often challenged and impeded by complex energy landscapes. To tackle the local trap problem that is typical in Metropolis sampling, the idea of dynamic weighting is incorporated into the parallel tempering (PT) algorithm. Our results show that, when applied to the lattice-protein model, the modified PT algorithm is capable of locating the low energy state much more quickly, but does not produce reliable estimates for equilibrium expectations. A modern method for free energy calculation, called the multistate Bennett acceptance ratio (MBAR) estimator, is reviewed from a statistical perspective, reminiscent of the underlying statistical theory which the method is based upon. Instead of adopting the common practice of using MBAR as a post-simulation analysis tool, we propose a new approach that integrates MBAR into simulation, allowing the simulation to benefit from the statistical optimality of the MBAR estimator. We show that the MBAR-enhanced Monte Carlo improves simulation efficiency of the lattice-protein aggregation model and, since it can also be applied to continuous models, provides a promising alternative to the study of more realistic systems. The new method is then applied to our model of TatA, where the protein features both a transmembrane and an amphipathic helix. The effect of individual helices on dimerization was studied and problem with the move set was identified. In this thesis, we used pull move and translation move as our Monte Carlo trial moves. Implementation details of pull moves, which are often omitted by many researchers who use them for sampling configuration space, are given in Chapter 1. We show that, for our double-helix TatA model, pull moves are no longer efficient moves and therefore, for future study of more realistic systems, we point to several methods which all attempt to design efficient trial moves. Aggregation of more than two polymer chains was also considered in this thesis

Monte Carlo simulation of DNA origami self-assembly

The optimal design of DNA origami systems that assemble rapidly and robustly is hampered by the lack of a model capable of simulating the self-assembly process that is sufficiently detailed yet computationally tractable. In this thesis, we propose a model for DNA origami that strikes a balance between these two criteria by representing DNA origami systems on a lattice at the level of binding domains. Particular attention is paid to the constraints imposed by the double-helical twist, as they determine where strand crossovers between adjacent helices can occur. Because of the highly specific types of interaction and the length of the scaffold, standard Monte Carlo simulation methods for polymeric systems are found to be ineffective at sampling the dense, near-assembled states considered here. In order to address the issue of sampling such states, we develop Monte Carlo methods that extend the configurational bias and recoil growth methods, and consider the sampling of scaffold conformations independently from the sampling of staple binding states. We demonstrate the validity of our model and the feasibility of our sampling methods with simulations of a small origami design previously studied with the oxDNA model, as well as with designs that include staples that span longer scaffold segments. In other self-assembling systems, it is often the case that nucleation barriers control the self-assembly behaviour. We investigate whether there is a nucleation barrier along the self-assembly pathway of DNA origami. Our simulations reveal that for simple systems, stacking interactions govern a nucleation barrier, albeit one that is never prohibitively large relative to thermal fluctuations. These findings may prove useful in the design of DNA origami structures capable of controllable reversible folding for functional purposes and in assisting the optimization of assembly pathways at the design stage

BIOLOGICAL AND ECOLOGICAL RESPONSES TO CARBON-BASED NANOMATERIALS

Nanotechnology has been undergoing tremendous development in recent decades, driven by realized perceived applications of nanomaterials in electronics, therapeutics, imaging, sensing, environmental remediation, and consumer products. Along with these developments there have been increased evidences that engineered nanomaterials are often associated with hazardous effects they invoke in biological and ecosystems through intentional designs or unintentional discharge. Consequently there is a crucial need for documenting and understanding the interactions between nanoparticles and biological and ecosystems. This dissertation is aimed at bridging such a knowledge gap by examining the biological and ecological responses to carbon nanoparticles, a major class of nanomaterials which have been mass produced and extensively studied for their rich physical properties and commercial values. Chapter I of this dissertation offers a comprehensive review on the structures, properties, applications, and implications of carbon nanomaterials, especially related to the perspectives of biological and ecosystems. Given that there are many types of carbon nanomaterials available, this chapter is focused on three major types of carbon-based nanomaterials only, namely, fullerenes, single walled and multi-walled carbon nanotubes. Based on the literature review in Chapter I, Chapters II-VI present step-by-step my Doctor of Philosophy (PhD) research on elucidating the biological and ecological responses to carbon nanoparticle exposure, from the whole organism level down to the cellular and molecular level. On the whole organism level, specifically, Chapter II presents a first study on the fate of fullerenes and multiwalled carbon nanotubes in rice plants, which was facilitated by the self assembly of these nanomaterials with NOM. The aspects of fullerene uptake, translocation, biodistribution, and generational transfer in the plants were examined and quantified using bright field and electron microscopy, FT-Raman, and FTIR spectroscopy. The uptake and transport of fullerene in the plant vascular system were attributed to water transpiration, convection, capillary force, and the fullerene concentration gradient from the roots to the leaves of the plants. On the cellular level, Chapter III documents the differential uptake of hydrophilic C60(OH)20 vs. amphiphilic C70-NOM complex in Allium cepa plant cells and HT-29 colon carcinoma cells. This study was conducted using a plant cell viability assay, and complemented by bright field, fluorescence and electron microscopy imaging. In particular, C60(OH)20 and C70-NOM showed contrasting uptake in both the plant and mammalian cells, due to their significant differences in physicochemistry and the presence of an extra hydrophobic plant cell wall in the plant cells. Consequently, C60(OH)20 was found to induce toxicity in Allium cepa cells but not in HT-29 cells, while C70-NOM was toxic to HT-29 cells but not to the plant cells. Along with the biophysical study presented in Chapter III, Chapter IV further delineates the toxicological consequences of cell exposure to C60(OH)20. The cytoprotective properties of C60(OH)20 against copper were demonstrated using a double-exposure system: HT-29 cells were first exposed to C60(OH)20 and then to copper, a physicologically essential element and a major toxin. Using cell viability, proliferation, and intracellular reactive oxygen species (ROS) production assays, I demonstrated the inhibition of copper-induced cell damage and ROS production by C60(OH)20. Neutralization of copper ions by C60(OH)20 in the extracellular space, as well as adsorption and uptake of the nanoparticles surface-modified by the cell medium were identified as plausible mechanisms for the cytoprotective activities of C60(OH)20 against copper. Extended from the cellular studies in Chapters III and IV, Chapter V and VI show molecular-level inhibitions of two major cellular processes -- DNA amplification and MT polymerization -- by C60(OH)20. Such inhibitions were mainly attributed to the formation of hydrogen bonding between the nanoparticles and the hydrogens of the triphosphate tail of the nucleotide/DNA or the tubulin heterodimers, the building blocks of microtubules. Specifically, in Chapter V, the effect of C60(OH)20 on the amplification of an HSTF gene was examined using PCR and real-time PCR, whereas in Chapter VI circular dichroism spectroscopy, GTP hydrolysis assay, and ITC measurements were utilized to examine the effect of C60(OH)20 on MT polymerization. In both cases, the experimental results were confirmed and substantiated by molecular dynamics simulations. Based on the studies documented in Chapters II-VI, Chapter VII summarizes and rationalizes the results obtained from the dissertation research and projects future work which may be beneficial to our understanding of nanoparticles at large. In short, this dissertation is composed of the following chapters: o Chapter I: Literature review o Chapter II: Uptake, translocation and transmission of carbon-based nanomaterials in rice plants o Chapter III: Differential uptake of carbon nanomaterials by plant and mammalian cells o Chapter IV: Cytoprotective properties of a fullerene derivative against copper o Chapter V: Experimental and simulations studies of a real-time PCR in the presence of a fullerene derivative o Chapter VI: In vitro polymerization of microtubules with a fullerene derivative o Chapter VII: Conclusions and future wor

Advances in Computational Solvation Thermodynamics

The aim of this thesis is to develop improved methods for calculating the free energy, entropy and enthalpy of solvation from molecular simulations. Solvation thermodynamics of model compounds provides quantitative measurements used to analyze the stability of protein conformations in aqueous milieus. Solvation free energies govern the favorability of the solvation process, while entropy and enthalpy decompositions give insight into the molecular mechanisms by which the process occurs. Computationally, a coupling parameter λ modulates solute-solvent interactions to simulate an insertion process, and multiple lengthy simulations at a fixed λ value are typically required for free energy calculations to converge; entropy and enthalpy decompositions generally take 10-100 times longer. This thesis presents three advances which accelerate the convergence of such calculations: 1) Development of entropy and enthalpy estimators which combine data from multiple simulations; 2) Optimization of λ schedules, or the set of parameter values associated with each simulation; 3) Validation of Hamiltonian replica exchange, a technique which swaps λ values between two otherwise independent simulations. Taken together, these techniques promise to increase the accuracy and precision of free energy, entropy and enthalpy calculations. Improved estimates, in turn, can be used to investigate the validity and limits of existing solvation models and refine force field parameters, with the goal of understanding better the collapse transition and aggregation behavior of polypeptides

Report / Institute für Physik

The 2015 Report of the Physics Institutes of the Universität Leipzig presents an interesting overview of our research activities in the past year. It is also testimony of our scientific interaction with colleagues and partners worldwide

Studies of the membrane influenza A/M2 protein with aminoadamantane drugs using experimental and computational biophysics

Chapter 1 refers to the description of the basic features for influenza A virus replication, with emphasis on the function of influenza A M2. In Chapter 2, is described the structure of influenza A matrix 2 (M2) wild type (WT) proton protein channel, which is an archetypal ion channel. It is the target of the antiviral drugs amantadine and rimantadine. A number of methods were used to understand structural and functional features of this channel included neutron diffraction, electrophysiology, solution NMR spectroscopy, solid state NMR (ssNMR) spectroscopy, X-ray crystallography etc during an adventure of three decades with a lot of controversies. The experimental structure of influenza A M2(22-46) transmembrane domain (M2TM), the pore of the M2 protein channel, was solved in 2000 and X-ray structures of its complexes with amantadine, rimantadine etc were published by 2018. Till now basic characteristics of the influenza A M2 conductance domain (CD) protein M2CD or M2AH including M2TM and the amphipathic helices (46-62) have been also solved using ssNMR.The basic features of the experimental biophysical methods used in this PhD thesis, i.e. Differential Scanning Calorimetry (DSC), X-ray scattering at small and wide angles (SAXS/WAXS) and ssNMR are discussed in Chapter 3. Aminoadamantane drugs, e.g. amantadine and rimantadine, are lipophilic amines that bind to membrane embedded influenza A WT M2 protein. In Chapter 4, are investigated the comparative perturbation effects exerted by the influenza M2 WT protein inhibitors amantadine and it’s spiro[pyrrolidine-2,2'-adamantane] variant AK13 to membrane bilayers using biophysical methods and molecular dynamics (MD) simulations. This is a work performed in close collaboration with Professor’s Thomas Mavromoustakos and Professor’s Costas Demetzos groups as well as Dr Barbara Sartori and Professor Heinz Amenitsch. The experimental biophysical methods used included, DSC, X-ray scattering and ssNMR. All three experimental methods pointed out that the two analogs perturbed drastically the DMPC bilayers with AK13 to be more effective at high concentrations. At high ligand concentrations AK13 was tolerated in lipid bilayers while Amt was crystallized. This is an important consideration in possible formulations of these drugs as it designates a limitation of aminoadamantane drug incorporation. MD simulations provided details about the strong interactions of the drugs in the interface region between glycerol backbone and lipophilic segments. The two drugs form hydrogen bonding with both water and sn-2 carbonyls or phosphate oxygens. Such localization of the drugs explains their strong perturbing effect evidenced by all biophysical methodologies applied.In Chapter 5, is described our work to investigate the interactions of M2TM WT with bilayers. This is a work performed in close collaboration with Professor Thomas Mavromoustakos, Professor Costas Demetzos, groups as well as Dr Barbara Sartori and Professor Heinz Amenitsch. The M2TM peptide was synthesized by Professor Thedoros Tselios group. We focused on (a) the characterization of changes in bilayer organization from changes in micromolar concentrations of M2TM WT without or with aminoadamantane (Aamt) ligands, and from changes in Aamt ligand structure included with M2TM, (b) exploring how common biophysical methods can be applied to identify the membrane perturbations effected by the protein without or with the ligand.A variety of biophysical methods, including DSC, SAXS/WAXS, MD simulations, and one-dimension (1D) ssNMR, were used to study two micromolar concentrations of M2TM without or with a small excess of amantadine or its spiro-pyrrolidine analogue, AK13, in DMPC bilayers.DSC and SAXS showed that at a low micromolar M2TM concentration, two lipid domains are observed, which likely correspond to M2TM boundary lipids and bulk lipids. At a higher M2TM concentration, only one domain is identified, indicating that all of the lipids behave as boundary lipids. 1H and 31P ssNMR showed that M2TM in either apo or drug-bound form spans the membrane, interacting strongly with lipid acyl chain-tails and the phosphate groups of the polar head surface. The 13C ssNMR experiments allowed the inspection of excess drug molecules and the assessment of their impact on the lipid head group region.According to SAXS, WAXS, and DSC, in the absence of M2TM both aminoadamantane drugs exert a similar perturbing effect on the bilayer at low concentrations, i.e., mole fractions (relative to lipid) of x=0.05-0.08. At the same concentrations of the drug when M2TM is present, the amantadine and, to a lesser extent, AK13 cause a significant disordering of chain-stacking. This different effect between the two drugs is likely due, according to the MD simulations, to the preference of the excess of the more lipophilic AK13 to locate closer to M2TM. In contrast, amantadine perturbs the lipids through the stronger ionic interactions of its ammonium group with phosphate groups (compared with the buried ammonium group in AK13) and influences the formation of two lipid domains. The preference of AK13 to concentrate inside the lipid may contribute to its six-fold higher binding affinity (compared to amantadine) if drug binding occurs from the lipid by way of a path between the transmembrane helices.The results showed that DSC and SAXS are useful methods to detect changes in membrane organization caused by small changes in M2TM or aminoadamantane drug concentration and structure and that WAXS and MD simulations can suggest details of ligand topology. Water-mediated interactions play key roles in drug binding. In protein sites with sparse polar functionality, a ligand-based only approach is often viewed as insufficient to achieve high affinity and specificity. In Chapter 6, are showed that small molecules, i.e. amantadine and rimantadine, can enable potent inhibition by targeting key waters using as example the M2 WT proton channel of influenza A which is the target of the antiviral drugs amantadine and rimantadine. This is a work performed in close collaboration with Professor William DeGrado and Associate Professor Jun Wang groups. Structural studies of drug binding to the channel using X-ray crystallography have been limited due to the challenging nature of the target, with the first crystal structure solved in 2008 limited to 3.5 Å resolution. We described crystal structures of amantadine bound to M2 in the Inwardclosed conformation (2.00 Å), rimantadine bound to M2 in both the Inwardclosed (2.00 Å) and Inwardopen (2.25 Å) conformations, and a spiro-adamantyl amine inhibitor bound to M2 in the Inwardclosed conformation (2.63 Å). These X-ray crystal structures of the M2 proton channel with bound inhibitors reveal that ammonium groups bind to water-lined sites, formed by two layers of waters close to Ala30 and Gly34, respectively, observed in the X-ray structures, that are hypothesized to stabilize transient hydronium ions formed in the proton-conduction mechanism. Furthermore, the ammonium and adamantyl groups of the adamantyl-amine class of drugs are free to rotate in the channel, minimizing the entropic cost of binding. The MD simulation reproduced perfectly the X-ray structures of cautiously tuned. These drug-bound complexes provide the first high-resolution structures of drugs that interact with and disrupt networks of hydrogen-bonded waters that are widely utilized throughout nature to facilitate proton diffusion within proteins.The V27A mutation confers amantadine resistance to the influenza A M2 WT proton channel and is becoming more prevalent in circulating populations of influenza A virus. In Chapter 7, is described our collaborative work with DeGrado and Wang groups to solve M2TM V27A structure in complex with a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation using X-ray crystallography and MD simulations. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 WT with valine at position 27, we observed that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in both the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to the M2(22-46) construct. We observed that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. However, the orientation of AHs after residue 48 did not reproduce the almost vertical orientation as regards the M2TM, that found by Professor Tim Cross experimentally with ssNMR experiments. The MD simulations of the M2(22-46) V27A - spiro-adamantyl amine complex predicted with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2 V27A complex.The influenza A M2 wild type proton channel is the target of the anti-influenza drug rimantadine. Rimantadine has two enantiomers, though most investigations into drug binding and inhibition have used a racemic mixture. ssNMR experiments by Professor Tim Cross have shown significant spectral differences that were interpreted to indicate tighter binding for (R)- vs. (S)- rimantadine. However, it was unclear if this is due to the specific condition of the ssNMR experiments (i.e. close to 0 oC), correlates with a functional difference in drug binding and inhibition and we undertook to investigate this in collaboration with Professor DeGrado, Associate Professor Jun Wang and Professor Jon Essex. Thus, in Chapter 8, using X-ray crystallography, we have determined that both (R)- and (S)-rimantadine bind to the M2 pore with slight differences in the hydration of each enantiomer. However, this did not result in a difference in potency or binding kinetics, as we measured similar values for kon, koff, and Kd in electrophysiological assays and EC50 values in cellular assays. We concluded that the slight differences in hydration we observed in the X-ray structures for the (R)- and (S)-rimantadine enantiomers were not relevant to drug binding or channel inhibition. To further explore the effect of the hydration of the M2 pore on binding affinity, the water structure was evaluated by waters titration calculations Grand Canonical Monte Carlo simulations as a function of the chemical potential of the water. Initially, the two layers of ordered water molecules between the bound drug and the channel's gating His37 residues mask the drug’s chirality. As the chemical potential becomes more unfavorable and the waters from the two layers were removed from the M2 pore, the drug translocated down to the lower water layer, towards the His37 at the C-terminus of M2TM, and the interaction becomes more sensitive to chirality. These studies suggested the feasibility of displacing the upper water layer (toward the N-end close to Ala30) and specifically recognizing the lower water layers by novel chiral drugs.Chapter 1 refers to the description of the basic features for influenza A virus replication, with emphasis on the function of influenza A M2. In Chapter 2, is described the structure of influenza A matrix 2 (M2) wild type (WT) proton protein channel, which is an archetypal ion channel. It is the target of the antiviral drugs amantadine and rimantadine. A number of methods were used to understand structural and functional features of this channel included neutron diffraction, electrophysiology, solution NMR spectroscopy, solid state NMR (ssNMR) spectroscopy, X-ray crystallography etc during an adventure of three decades with a lot of controversies. The experimental structure of influenza A M2(22-46) transmembrane domain (M2TM), the pore of the M2 protein channel, was solved in 2000 and X-ray structures of its complexes with amantadine, rimantadine etc were published by 2018. Till now basic characteristics of the influenza A M2 conductance domain (CD) protein M2CD or M2AH including M2TM and the amphipathic helices (46-62) have been also solved using ssNMR.The basic features of the experimental biophysical methods used in this PhD thesis, i.e. Differential Scanning Calorimetry (DSC), X-ray scattering at small and wide angles (SAXS/WAXS) and ssNMR are discussed in Chapter 3. Aminoadamantane drugs, e.g. amantadine and rimantadine, are lipophilic amines that bind to membrane embedded influenza A WT M2 protein. In Chapter 4, are investigated the comparative perturbation effects exerted by the influenza M2 WT protein inhibitors amantadine and it’s spiro[pyrrolidine-2,2'-adamantane] variant AK13 to membrane bilayers using biophysical methods and molecular dynamics (MD) simulations. This is a work performed in close collaboration with Professor’s Thomas Mavromoustakos and Professor’s Costas Demetzos groups as well as Dr Barbara Sartori and Professor Heinz Amenitsch. The experimental biophysical methods used included, DSC, X-ray scattering and ssNMR. All three experimental methods pointed out that the two analogs perturbed drastically the DMPC bilayers with AK13 to be more effective at high concentrations. At high ligand concentrations AK13 was tolerated in lipid bilayers while Amt was crystallized. This is an important consideration in possible formulations of these drugs as it designates a limitation of aminoadamantane drug incorporation. MD simulations provided details about the strong interactions of the drugs in the interface region between glycerol backbone and lipophilic segments. The two drugs form hydrogen bonding with both water and sn-2 carbonyls or phosphate oxygens. Such localization of the drugs explains their strong perturbing effect evidenced by all biophysical methodologies applied.In Chapter 5, is described our work to investigate the interactions of M2TM WT with bilayers. This is a work performed in close collaboration with Professor Thomas Mavromoustakos, Professor Costas Demetzos, groups as well as Dr Barbara Sartori and Professor Heinz Amenitsch. The M2TM peptide was synthesized by Professor Thedoros Tselios group. We focused on (a) the characterization of changes in bilayer organization from changes in micromolar concentrations of M2TM WT without or with aminoadamantane (Aamt) ligands, and from changes in Aamt ligand structure included with M2TM, (b) exploring how common biophysical methods can be applied to identify the membrane perturbations effected by the protein without or with the ligand.A variety of biophysical methods, including DSC, SAXS/WAXS, MD simulations, and one-dimension (1D) ssNMR, were used to study two micromolar concentrations of M2TM without or with a small excess of amantadine or its spiro-pyrrolidine analogue, AK13, in DMPC bilayers.DSC and SAXS showed that at a low micromolar M2TM concentration, two lipid domains are observed, which likely correspond to M2TM boundary lipids and bulk lipids. At a higher M2TM concentration, only one domain is identified, indicating that all of the lipids behave as boundary lipids. 1H and 31P ssNMR showed that M2TM in either apo or drug-bound form spans the membrane, interacting strongly with lipid acyl chain-tails and the phosphate groups of the polar head surface. The 13C ssNMR experiments allowed the inspection of excess drug molecules and the assessment of their impact on the lipid head group region.According to SAXS, WAXS, and DSC, in the absence of M2TM both aminoadamantane drugs exert a similar perturbing effect on the bilayer at low concentrations, i.e., mole fractions (relative to lipid) of x=0.05-0.08. At the same concentrations of the drug when M2TM is present, the amantadine and, to a lesser extent, AK13 cause a significant disordering of chain-stacking. This different effect between the two drugs is likely due, according to the MD simulations, to the preference of the excess of the more lipophilic AK13 to locate closer to M2TM. In contrast, amantadine perturbs the lipids through the stronger ionic interactions of its ammonium group with phosphate groups (compared with the buried ammonium group in AK13) and influences the formation of two lipid domains. The preference of AK13 to concentrate inside the lipid may contribute to its six-fold higher binding affinity (compared to amantadine) if drug binding occurs from the lipid by way of a path between the transmembrane helices.The results showed that DSC and SAXS are useful methods to detect changes in membrane organization caused by small changes in M2TM or aminoadamantane drug concentration and structure and that WAXS and MD simulations can suggest details of ligand topology. Water-mediated interactions play key roles in drug binding. In protein sites with sparse polar functionality, a ligand-based only approach is often viewed as insufficient to achieve high affinity and specificity. In Chapter 6, are showed that small molecules, i.e. amantadine and rimantadine, can enable potent inhibition by targeting key waters using as example the M2 WT proton channel of influenza A which is the target of the antiviral drugs amantadine and rimantadine. This is a work performed in close collaboration with Professor William DeGrado and Associate Professor Jun Wang groups. Structural studies of drug binding to the channel using X-ray crystallography have been limited due to the challenging nature of the target, with the first crystal structure solved in 2008 limited to 3.5 Å resolution. We described crystal structures of amantadine bound to M2 in the Inwardclosed conformation (2.00 Å), rimantadine bound to M2 in both the Inwardclosed (2.00 Å) and Inwardopen (2.25 Å) conformations, and a spiro-adamantyl amine inhibitor bound to M2 in the Inwardclosed conformation (2.63 Å). These X-ray crystal structures of the M2 proton channel with bound inhibitors reveal that ammonium groups bind to water-lined sites, formed by two layers of waters close to Ala30 and Gly34, respectively, observed in the X-ray structures, that are hypothesized to stabilize transient hydronium ions formed in the proton-conduction mechanism. Furthermore, the ammonium and adamantyl groups of the adamantyl-amine class of drugs are free to rotate in the channel, minimizing the entropic cost of binding. The MD simulation reproduced perfectly the X-ray structures of cautiously tuned. These drug-bound complexes provide the first high-resolution structures of drugs that interact with and disrupt networks of hydrogen-bonded waters that are widely utilized throughout nature to facilitate proton diffusion within proteins.The V27A mutation confers amantadine resistance to the influenza A M2 WT proton channel and is becoming more prevalent in circulating populations of influenza A virus. In Chapter 7, is described our collaborative work with DeGrado and Wang groups to solve M2TM V27A structure in complex with a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation using X-ray crystallography and MD simulations. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 WT with valine at position 27, we observed that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in both the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to the M2(22-46) construct. We observed that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. However, the orientation of AHs after residue 48 did not reproduce the almost vertical orientation as regards the M2TM, that found by Professor Tim Cross experimentally with ssNMR experiments. The MD simulations of the M2(22-46) V27A - spiro-adamantyl amine complex predicted with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2 V27A complex.The influenza A M2 wild type proton channel is the target of the anti-influenza drug rimantadine. Rimantadine has two enantiomers, though most investigations into drug binding and inhibition have used a racemic mixture. ssNMR experiments by Professor Tim Cross have shown significant spectral differences that were interpreted to indicate tighter binding for (R)- vs. (S)- rimantadine. However, it was unclear if this is due to the specific condition of the ssNMR experiments (i.e. close to 0 oC), correlates with a functional difference in drug binding and inhibition and we undertook to investigate this in collaboration with Professor DeGrado, Associate Professor Jun Wang and Professor Jon Essex. Thus, in Chapter 8, using X-ray crystallography, we have determined that both (R)- and (S)-rimantadine bind to the M2 pore with slight differences in the hydration of each enantiomer. However, this did not result in a difference in potency or binding kinetics, as we measured similar values for kon, koff, and Kd in electrophysiological assays and EC50 values in cellular assays. We concluded that the slight differences in hyd

Výpočetní studie krátkých peptidů a miniproteinů a vliv prostředí na jejich konformaci.

Apart from biological functions, peptides are of uttermost importance as models for un- folded, denatured or disordered state of the proteins. Similarly, miniproteins such as Trp-cage have proven their role as simple models of both experimental and theoretical studies of protein folding. Molecular dynamics and computer simulations can provide an unique insight on processes at atomic level. However, simulations of peptides and minipro- teins face two cardinal problems-inaccuracy of force fields and inadequate conformation sampling. Both principal issues were tackled in this theses. Firstly, the differences in several force field for peptides and proteins were questioned. We demonstrated the inability of the used force fields to predict consistently intrinsic conformational preferences of individual amino acids in the form of dipeptides and the source of the discrepancies was traced. In order to shed light on the nature of conformational ensembles under various denatur- ing conditions, we studied host-guest AAXAA peptides. The simulations revealed that thermal and chemical denaturation by urea produces qualitatively different ensembles and shift propensities of individual amino acids to particular conformers. The problem of insufficient conformation sampling was dealt by introducing gyration- and...Peptidy, kromě své biologické funkce, představují take důležité modely nesbalených, de- naturovaných nebo nestrukturovaných proteinů. Pobobně důležitými modely pro exper- imentální i teoretické studium sbalování proteinů jsou miniproteiny, jako např. Trp- cage. Chování peptidů i proteinů lze studovat v počítačových simulacích pomocí metod molekulární dynamiky, které umožnují sledovat děje v atomistickém rozlišení. Tyto metody však čelí však dvěma zásadním problémům - přesnosti používaných energetick- ých funkcí a nedostatečnému vzorkování konformačních stavů. V této disertaci jsem se zabýval oběma okruhy problémů. Vliv rozdílných, běžně používných energetických funkcí ("force fields") byl testován na modelu aminokyselinových dipeptidů. Žádná sada parametrů však nedokázala konzis- tentně reprodukovat konformační preference jednotlivých aminokyselin. Výsledky simu- lací byly mezi sebou srovnány a byly hledány příčiny jejich vzájemných odlišností. Abychom odhalili, jakým způsobem různé podmínky ovlivňují konformační stavy peptidů, zkoumali jsme vlastnosti aminokyselin v AAXAA peptidech. Simulace odhalily zásadní rozdíl ve vlivu tepelné a chemické denaturace (močovinou) na charakter a zastoupení konformací peptidů, stejně jako konformačních preferencí jednotlivých aminokyselin. K problematice vzorkování...Department of Physical and Macromolecular ChemistryKatedra fyzikální a makromol. chemieFaculty of SciencePřírodovědecká fakult

Next-generation coarse-grained models for molecular dynamics simulations of fluid phase equilibria and protein biophysics using the relative entropy

Coarse grained models for molecular dynamic simulations of liquid structure and protein folding and self-assembly have been the subject of decades of research efforts. Although such models enable probing into longer length and time scales, they are still limited in accuracy and scale poorly to thermodynamic conditions or chemical entities beyond the ones at which they are developed. In this work, I leverage a powerful coarse graining framework based on an information theoretic metric known as the relative entropy to design novel coarse grained models for equilibrium phase behavior in liquid mixtures that correctly address the relevant manybody physics critically involved in phase behavior and thus, can provide structurally accurate descriptions of macroscopic phase separation across a large range of mixture compositions. I also develop protein models that are “next- generation” in the sense that they are systematically extendable in complexity while depending minimally on experimental data. These protein models offer remarkable predictive accuracy for folding of single proteins and insights into large scale protein self-assembly commonly seen in neurodegenerative pathologies such as Alzheimer’s and prion diseases. This dissertation develops and applies powerful coarse-graining techniques to meet longstanding challenges in the field and elevate coarse grained models from being “toy” systems to accurate, “production-level” tools for the development of biotechnologies and advanced materials