Jochen S. Hub

Molecular Anatomy of a Trafficking Organelle

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.

Mechanism of selectivity in aquaporins and aquaglyceroporins

Aquaporins and aquaglyceroporins form a family of pore proteins that facilitate the efficient and selective flux of small solutes across biological membranes. We studied the selectivity of aquaporin-1 (AQP1) and the bacterial glycerol facilitator, GlpF, for O2, CO2, NH3, glycerol, urea, and water. Using molecular dynamics simulations, we calculated potentials of mean force for solute permeation along the aquaporin channels and compared them with the alternative pathway across the lipid bilayer. For small solutes permeating through AQP1, a remarkable anticorrelation between permeability and solute hydrophobicity was observed, whereas the opposite trend was observed for permeation through the membrane. This finding renders AQP1 a selective filter for small polar solutes, whereas GlpF was found to be highly permeable for small solutes and permeable for larger solutes. Surprisingly, not solute-channel but water-channel interactions were found to be the key determinant underlying the selectivity mechanism of aquaporins. Hence, a hydrophobic effect, together with steric restraints, determines the selectivity of aquaporins.

Atomistic simulation of ion solvation in water explains surface preference of halides

Water is a demanding partner. It strongly attracts ions, yet some halide anions—chloride, bromide, and iodide—are expelled to the air/water interface. This has important implications for chemistry in the atmosphere, including the ozone cycle. We present a quantitative analysis of the energetics of ion solvation based on molecular simulations of all stable alkali and halide ions in water droplets. The potentials of mean force for Cl-, Br-, and I- have shallow minima near the surface. We demonstrate that these minima derive from more favorable water–water interaction energy when the ions are partially desolvated. Alkali cations are on the inside because of the favorable ion–water energy, whereas F- is driven inside by entropy. Models attempting to explain the surface preference based on one or more ion properties such as polarizability or size are shown to lead to qualitative and quantitative errors, prompting a paradigm shift in chemistry away from such simplifications.

Large Influence of Cholesterol on Solute Partitioning into Lipid Membranes

Cholesterol plays an important role in maintaining the correct fluidity and rigidity of the plasma membrane of all animal cells, and hence, it is present in concentrations ranging from 20 to 50 mol %. Whereas the effect of cholesterol on such mechanical properties has been studied exhaustively over the last decades, the structural basis for cholesterol effects on membrane permeability is still unclear. Here we apply systematic molecular dynamics simulations to study the partitioning of solutes between water and membranes. We derive potentials of mean force for six different solutes permeating across 20 different lipid membranes containing one out of four types of phospholipids plus a cholesterol content varying from 0 to 50 mol %. Surprisingly, cholesterol decreases solute partitioning into the lipid tail region of the membranes much more strongly than expected from experiments on macroscopic membranes, suggesting that a laterally inhomogeneous cholesterol concentration and permeability may be required to explain experimental findings. The simulations indicate that the cost of breaking van der Waals interactions between the lipid tails of cholesterol-containing membranes account for the reduced partitioning rather than the surface area per phospholipid, which has been frequently suggested as a determinant for solute partitioning. The simulations further show that the partitioning is more sensitive to cholesterol (i) for larger solutes, (ii) in membranes with saturated as compared to membranes with unsaturated lipid tails, and (iii) in membranes with smaller lipid head groups.

Detection of Functional Modes in Protein Dynamics

Proteins frequently accomplish their biological function by collective atomic motions. Yet the identification of collective motions related to a specific protein function from, e.g., a molecular dynamics trajectory is often non-trivial. Here, we propose a novel technique termed “functional mode analysis” that aims to detect the collective motion that is directly related to a particular protein function. Based on an ensemble of structures, together with an arbitrary “functional quantity” that quantifies the functional state of the protein, the technique detects the collective motion that is maximally correlated to the functional quantity. The functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. In addition, the motion that displays the largest likelihood to induce a substantial change in the functional quantity is estimated from the given protein ensemble. Two different correlation measures are applied: first, the Pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. Detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. The new approach is illustrated using a number of biomolecules, including a polyalanine-helix, T4 lysozyme, Trp-cage, and leucine-binding protein.

Potentials of Mean Force and Permeabilities for Carbon Dioxide, Ammonia, and Water Flux across a Rhesus Protein Channel and Lipid Membranes

As a member of the ubiquitous ammonium transporter/methylamine permease/Rhesus (Amt/MEP/Rh) family of membrane protein channels, the 50 kDa Rhesus channel (Rh50) has been implicated in ammonia (NH(3)) and, more recently, also in carbon dioxide (CO(2)) transport. Here we present molecular dynamics simulations of spontaneous full permeation events of ammonia and carbon dioxide across Rh50 from Nitrosomonas europaea. The simulations show that Rh50 is functional in its crystallographic conformation, without the requirement for a major conformational change or the action of a protein partner. To assess the physiological relevance of NH(3) and CO(2) permeation across Rh50, we have computed potentials of mean force (PMFs) and permeabilities for NH(3) and CO(2) flux across Rh50 and compare them to permeation through a wide range of lipid membranes, either composed of pure lipids or composed of lipids plus an increasing cholesterol content. According to the PMFs, Rh50 is expected to enhance NH(3) flux across dense membranes, such as membranes with a substantial cholesterol content. Although cholesterol reduces the intrinsic CO(2) permeability of lipid membranes, the CO(2) permeabilities of all membranes studied here are too high to allow significant Rh50-mediated CO(2) flux. The increased barrier in the PMF for water permeation across Rh50 shows that Rh50 discriminates 40-fold between water and NH(3). Thus, Rh50 channels complement aquaporins, allowing the cell to regulate water and NH(3) flux independently. The PMFs for methylamine and NH(3) are virtually identical, suggesting that methylamine provides an excellent model for NH(3) in functional experiments.

Quantifying artifacts in Ewald simulations of inhomogeneous systems with a net charge.

Ewald summation, which has become the de facto standard for computing electrostatic interactions in biomolecular simulations, formally requires that the simulation box is neutral. For non-neutral systems, the Ewald algorithm implicitly introduces a uniform background charge distribution that effectively neutralizes the simulation box. Because a uniform distribution of counter charges typically deviates from the spatial distribution of counterions in real systems, artifacts may arise, in particular in systems with an inhomogeneous dielectric constant. Here, we derive an analytical expression for the effect of using an implicit background charge instead of explicit counterions, on the chemical potential of ions in heterogeneous systems, which (i) provides a quantitative criterium for deciding if the background charge offers an acceptable trade-off between artifacts arising from sampling problems and artifacts arising from the homogeneous background charge distribution, and (ii) can be used to correct this artifact in certain cases. Our model quantifies the artifact in terms of the difference in charge density between the non-neutral system with a uniform neutralizing background charge and the real neutral system with a physically correct distribution of explicit counterions. We show that for inhomogeneous systems, such as proteins and membranes in water, the artifact manifests itself by an overstabilization of ions inside the lower dielectric by tens to even hundreds kilojoules per mole. We have tested the accuracy of our model in molecular dynamics simulations and found that the error in the calculated free energy for moving a test charge from water into hexadecane, at different net charges of the system and different simulation box sizes, is correctly predicted by the model. The calculations further confirm that the incorrect distribution of counter charges in the simulation box is solely responsible for the errors in the PMFs.

Validating Solution Ensembles from Molecular Dynamics Simulation by Wide-Angle X-ray Scattering Data

Wide-angle x-ray scattering (WAXS) experiments of biomolecules in solution have become increasingly popular because of technical advances in light sources and detectors. However, the structural interpretation of WAXS profiles is problematic, partly because accurate calculations of WAXS profiles from structural models have remained challenging. In this work, we present the calculation of WAXS profiles from explicit-solvent molecular dynamics (MD) simulations of five different proteins. Using only a single fitting parameter that accounts for experimental uncertainties because of the buffer subtraction and dark currents, we find excellent agreement to experimental profiles both at small and wide angles. Because explicit solvation eliminates free parameters associated with the solvation layer or the excluded solvent, which would require fitting to experimental data, we minimize the risk of overfitting. We further find that the influence from water models and protein force fields on calculated profiles are insignificant up to q≈15nm(-1). Using a series of simulations that allow increasing flexibility of the proteins, we show that incorporating thermal fluctuations into the calculations significantly improves agreement with experimental data, demonstrating the importance of protein dynamics in the interpretation of WAXS profiles. In addition, free MD simulations up to one microsecond suggest that the calculated profiles are highly sensitive with respect to minor conformational rearrangements of proteins, such as an increased flexibility of a loop or an increase of the radius of gyration by < 1%. The present study suggests that quantitative comparison between MD simulations and experimental WAXS profiles emerges as an accurate tool to validate solution ensembles of biomolecules.

WAXSiS: a web server for the calculation of SAXS/WAXS curves based on explicit-solvent molecular dynamics

Small- and wide-angle X-ray scattering (SWAXS) has evolved into a powerful tool to study biological macromolecules in solution. The interpretation of SWAXS curves requires their accurate predictions from structural models. Such predictions are complicated by scattering contributions from the hydration layer and by effects from thermal fluctuations. Here, we describe the new web server WAXSiS (WAXS in solvent) that computes SWAXS curves based on explicit-solvent all-atom molecular dynamics (MD) simulations (http://waxsis.uni-goettingen.de/). The MD simulations provide a realistic model for both the hydration layer and the excluded solvent, thereby avoiding any solvent-related fitting parameters, while naturally accounting for thermal fluctuations.

Spontaneous quaternary and tertiary T-R transitions of human hemoglobin in molecular dynamics simulation.

We present molecular dynamics simulations of unliganded human hemoglobin (Hb) A under physiological conditions, starting from the R, R2, and T state. The simulations were carried out with protonated and deprotonated HC3 histidines His(β)146, and they sum up to a total length of 5.6µs. We observe spontaneous and reproducible T→R quaternary transitions of the Hb tetramer and tertiary transitions of the α and β subunits, as detected from principal component projections, from an RMSD measure, and from rigid body rotation analysis. The simulations reveal a marked asymmetry between the α and β subunits. Using the mutual information as correlation measure, we find that the β subunits are substantially more strongly linked to the quaternary transition than the α subunits. In addition, the tertiary populations of the α and β subunits differ substantially, with the β subunits showing a tendency towards R, and the α subunits showing a tendency towards T. Based on the simulation results, we present a transition pathway for coupled quaternary and tertiary transitions between the R and T conformations of Hb.