Exploring the activation pathway and Gi-coupling specificity of the µ-opioid receptor.

Understanding the activation mechanism of the µ-opioid receptor (µ-OR) and its selective coupling to the inhibitory G protein (Gi) is vital for pharmaceutical research aimed at finding treatments for the opioid overdose crisis. Many attempts have been made to understand the mechanism of the µ-OR activation, following the elucidation of new crystal structures such as the antagonist- and agonist-bound µ-OR. However, the focus has not been placed on the underlying energetics and specificity of the activation process. An energy-based picture would not only help to explain this coupling but also help to explore why other possible options are not common. For example, one would like to understand why µ-OR is more selective to Gi than a stimulatory G protein (Gs). Our study used homology modeling and a coarse-grained model to generate all of the possible "end states" of the thermodynamic cycle of the activation of µ-OR. The end points were further used to generate reasonable intermediate structures of the receptor and the Gi to calculate two-dimensional free energy landscapes. The results of the landscape calculations helped to propose a plausible sequence of conformational changes in the µ-OR and Gi system and for exploring the path that leads to its activation. Furthermore, in silico alanine scanning calculations of the last 21 residues of the C terminals of Gi and Gs were performed to shed light on the selective binding of Gi to µ-OR. Overall, the present work appears to demonstrate the potential of multiscale modeling in exploring the action of G protein-coupled receptors.

Combinatorial Approach for Exploring Conformational Space and Activation Barriers in Computer-Aided Enzyme Design.

Computer-aided enzyme design is a field of great potential importance for biotechnological applications, medical advances, and a fundamental understanding of enzyme action. However, reaching a predictive ability in this direction is extremely challenging. It requires both the ability to predict quantitatively the activation barriers in cases where the structure and sequence are known and the ability to predict the effect of different mutations. In this work, we propose a protocol for predicting reasonable starting structures of mutants of proteins with known structures and for calculating the activation barriers of the generated mutants. Our approach also allows us to use the predicted structures of the generated mutant to predict structures and activation barriers for subsequent set of mutations. This protocol is used to examine the reliability of the in silico directed evolution of Kemp eliminase and haloalkane dehalogenase. We also used the results of single and double mutations as a base for predicting the effect of transition-state stabilization by multiple concurrent mutations. This strategy seems to be useful in creating an activity funnel that provides a qualitative ranking of the catalytic power of different mutants.

Validating a Coarse-Grained Voltage Activation Model by Comparing Its Performance to the Results of Monte Carlo Simulations.

Simulating the nature of voltage-activated systems is a problem of major current interest, ranging from the action of voltage-gated ion channels to energy storage batteries. However, fully microscopic converging molecular simulations of external voltage effects present a major challenge, and macroscopic models are associated with major uncertainties about the dielectric treatment and the underlying physical basis. Recently we developed a coarse-grained (CG) model that represents explicitly the electrodes, the electrolytes, and the membrane/protein system. The CG model provides a semimacroscopic way of capturing the microscopic physics of voltage-activated systems. Our method was originally validated by reproducing macroscopic and analytical results for key test cases and then used in modeling voltage-activated ion channels and related problems. In this work, we further establish the reliability of the CG voltage model by comparing it to the results of Monte Carlo (MC) simulations with a microscopic electrolyte model. The comparison explores different aspects of membrane, electrolyte, and electrode systems ranging from the Gouy-Chapman model to the determination of the electrolyte charge distribution in the solution between two electrodes (without and with a separating membrane), as well as the evaluation of gating charges. Overall the agreement is very impressive. This provides confidence in the CG model and also shows that the MC model can be used in realistic simulation of voltage activation of membrane proteins with sufficient computer time.

Validating the Water Flooding Approach by Comparing It to Grand Canonical Monte Carlo Simulations.

The study of the function of proteins on a quantitative level requires consideration of the water molecules in and around the protein. This requirement presents a major computational challenge due to the fact that the insertion of water molecules can have a very high activation barrier and would require a long simulation time. Recently, we developed a water flooding (WF) approach which is based on a postprocessing Monte Carlo ranking of possible water configurations. This approach appears to provide a very effective way for assessing the insertion free energies and determining the most likely configurations of the internal water molecules. Although the WF approach was used effectively in modeling challenging systems that have not been addressed reliably by other microscopic approaches, it was not validated by a comparison to the more rigorous grand canonical Monte Carlo (GCMC) method. Here we validate the WF approach by comparing its performance to that of the GCMC method. It is found that the WF approach reproduces the GCMC results in well-defined test cases but does so much faster. This established the WF approach as a useful strategy for finding correct water configurations in proteins and thus to provide a powerful way for studies of the functions of proteins.

Unit cell structure of water-filled monoolein in inverted hexagonal mesophase in the presence of incorporated tricaprylin and entrapped lysozyme.

Molecular dynamics (MD) was employed by means of a specific simulation protocol to investigate the equilibrium structure at 25 °C of the hexagonal inverted (HII) mesophase composed from water, 1-monoolein (GMO), and tricaprylin, with or without entrapped lysozyme. Based on robust and fast MD simulations, the study provides a comprehensive analysis and visualization of the local structure of HII mesophase containing admixtures. The most important physical insight is the possibility to observe the strong self-recovery capacity of the GMO layer, which allows the HII mesophase tubes to reorganize and host lysozyme molecules with a size bigger than the diameter of the water channel. This is a direct message to the experimenters that the HII mesophase has the potential to host molecules larger than the diameter of the water channel. Collective character of the interlipid interactions is outlined, which is not affected by the presence of the cargo and may be the reason for the efficient GMO reorganization. Another important result is the possible explanation of the role of triacylglycerols on the low-temperature stabilization of the HII mesophase. The analysis shows that despite the low amount of tricaprylin, its molecules prevent the extreme inclination of the lipid tails and thus optimize the alignment capacity of the lipid tails layer. The study also reveals that the packing frustration does not depend on the temperature and the presence of admixtures. Hence, it might be numerically defined as a universal invariant parameter of a stable HII mesophase composed of a certain lipid.

Molecular Dynamics Investigation of Ion Sorption and Permeation in Desalination Membranes.

With the purpose of gaining insights into the mechanisms of ion uptake and permeation in desalination membranes, MD investigation of a model polyamide membrane was carried out. A relatively large membrane (45K atoms) was assembled, which closely matched real desalination membrane in terms of chemistry and water permeability. Simulations demonstrate that the mechanism of ion uptake distinctly differs from mean-field approaches assuming a smeared excluding Donnan potential. Ion sorption on charged sites in the membrane phase appears to be highly localized, due to electrostatic forces dominating over translational entropy. Moreover, sorption on partial atomic charges becomes possible as well, which greatly enhances salt (co-ion) uptake and weakens the effect of fixed charges on salt exclusion. This could explain high ion uptake measured in polyamide membranes for both co- and counterions and variations of ion sorption and permeation at low salt concentrations. On the other hand, present simulations greatly overestimate ion permeability, which could be explained by a more open structure than in real membranes, in which dense polyamide fragments may efficiently block ion permeation. Unfortunately, MD cannot analyze ion uptake and permeation in dense fragments containing too few ions, which calls for new approaches to studying barrier properties of polyamide.

Does hindered transport theory apply to desalination membranes?

As reverse osmosis (RO) and nanofiltration polyamide membranes become increasingly used for water purification, prediction of pollutant transport is required for membrane development and process engineering. Many popular models use hindered transport theory (HTT), which considers a spherical solute moving through an array of fluid-filled rigid cylindrical pores. Experiments and molecular dynamic simulations, however, reveal that polyamide membranes have a distinctly different structure of a "molecular sponge", a network of randomly connected voids widely distributed in size. In view of this disagreement, this study critically examined the validity of HTT by directly measuring diffusivities of several alcohols within a polyamide film of commercial RO membrane using attenuated total reflection-FTIR. It is found that measured diffusivities deviate from HTT predictions by as much as 2-3 orders of magnitude. This result indicates that HTT does not adequately describe solute transport in desalination membranes. As a more adequate alternative, the concept of random resistor networks is suggested, with resistances described by models of activated transport in "soft" polymers without a sharp size cutoff and with a proper address of solute partitioning.

Unit cell structure of water-filled monoolein into inverted hexagonal (H(II)) mesophase modeled by molecular dynamics.

The study investigates the unit cell structure of inverted hexagonal (H(II)) mesophase composed of monoolein (1-monoolein, GMO) and water using atomistic molecular dynamics methods without imposing any restraints on lipid and water molecules. Statistically meaningful and very contrast images of the radial mass density distribution, scrutinizing also the separate components water, monoolein, the polar headgroups of the lipids, the double bond, and the termini of the hydrocarbon chain (the tail), are obtained. The lipid/water interface structure is analyzed based on the obtained water density distribution, on the estimated number of hydrogen bonds per monoolein headgroup, and on the headgroup-water radial distribution functions. The headgroup mass density distribution demonstrates hexagonal shape of the monoolein/water interface that is well-defined at higher water/monoolein ratios. Water interacts with the headgroups by forming a three-layer diffusive mass density distribution, and each layer's shape is close to hexagonal, which is an indication of long-range structural interactions. It is found that the monoolein headgroups form a constant number of hydrogen bonds leaving an excessive amount of water molecules outside the first lipid coordination sphere. Furthermore, the quantity of water at the monoolein/water interface increases steadily upon extension of the unit cell, so the interface should have a very dynamic structure. Investigation of the hydrocarbon residues reveals high compression and well-expressed structuring of the tails. The tails form a very compressed and constrained structure of defined layers across the unit cell with properties corresponding to a more densely packed nonpolar liquid (oil). Due to the hexagonal shape the 2D packing frustration is constant and does not depend on the water content. All reported structural features are based on averaging of the atomic coordinates over the time-length of the simulation trajectories. That kind of processing allows the observation of the water/GMO interface shape and its stability and mobility at a time scale close to the ones of the intermolecular interactions.

Molecular dynamics approach to water structure of H(II) mesophase of monoolein.

The goal of the present work is to study theoretically the structure of water inside the water cylinder of the inverse hexagonal mesophase (H(II)) of glyceryl monooleate (monoolein, GMO), using the method of molecular dynamics. To simplify the computational model, a fixed structure of the GMO tube is maintained. The non-standard cylindrical geometry of the system required the development and application of a novel method for obtaining the starting distribution of water molecules. A predictor-corrector schema is employed for generation of the initial density of water. Molecular dynamics calculations are performed at constant volume and temperature (NVT ensemble) with 1D periodic boundary conditions applied. During the simulations the lipid structure is kept fixed, while the dynamics of water is unrestrained. Distribution of hydrogen bonds and density as well as radial distribution of water molecules across the water cylinder show the presence of water structure deep in the cylinder (about 6 Å below the GMO heads). The obtained results may help understanding the role of water structure in the processes of insertion of external molecules inside the GMO∕water system. The present work has a semi-quantitative character and it should be considered as the initial stage of more comprehensive future theoretical studies.

Generation of tuneable 589nm radiation as a Na guide star source using an optical parametric amplifier.

We describe a 5.5W 589 nm source based on a passively mode-locked Nd:YVO4 laser and a multi-stage Lithium Triborate optical parametric amplifier seeded by a tuneable semiconductor laser. We show this system can produce rapidly tuneable, transform-limited pulses in near diffraction-limited beams at 589 nm, useful for Na guide star applications. The attraction of this scheme is that it can be assembled from commercially available hardware and is readily scalable to high average powers.

Nonlinear dynamics of two-color optical vortices in lithium niobate crystals.

We study experimentally the nonlinear dynamics of two-color optical vortex beams in the presence of second-harmonic generation combined with the effects of photo- and thermal refraction, as well as self- and induced-phase modulation. We use an iron-doped lithium niobate crystal as a nonlinear medium for the vortex propagation and observe experimentally, depending on the laser wavelength, a decay of a double-charge vortex, splitting and reshaping of background beam, pattern formation, and controllable nonlinear rotation of a vortex pair.