Insights into receptor structure and dynamics at the surface of living cells.

Evaluating protein structures in living cells remains a challenge. Here, we investigate Interleukin-4 receptor alpha (IL-4Rα) into which the non-canonical amino acid bicyclo[6.1.0]nonyne-lysine (BCNK) is incorporated by genetic code expansion. Bioorthogonal click labeling is performed with tetrazine-conjugated dyes. To quantify the reaction yield in situ, we develop brightness-calibrated ratiometric imaging, a protocol where fluorescent signals in confocal multi-color images are ascribed to local concentrations. Screening receptor mutants bearing BCNK in the extracellular domain uncovered site-specific variations of both click efficiency and Interleukin-4 binding affinity, indicating subtle well-defined structural perturbations. Molecular dynamics and continuum electrostatics calculations suggest solvent polarization to determine site-specific variations of BCNK reactivity. Strikingly, signatures of differential click efficiency, measured for IL-4Rα in ligand-bound and free form, mirror sub-angstrom deformations of the protein backbone at corresponding locations. Thus, click efficiency by itself represents a remarkably informative readout linked to protein structure and dynamics in the native plasma membrane.

Impact of the green tea ingredient epigallocatechin gallate and a short pentapeptide (Ile-Ile-Ala-Glu-Lys) on the structural organization of mixed micelles and the related uptake of cholesterol.

BACKGROUND: High levels of blood cholesterol are conventionally linked to an increased risk of developing cardiovascular disease (Grundy, 1986). Here we examine the molecular mode of action of natural products with known cholesterol-lowering activity, such as for example the green tea ingredient epigallocatechin gallate and a short pentapeptide, Ile-Ile-Ala-Glu-Lys. METHODS: Molecular Dynamics simulations are used to gain insight into the formation process of mixed micelles and, correspondingly, how active agents epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys could possibly interfere with it. RESULTS: Self-assembly of physiological micelles occurs on the order of 35-50 ns; most of the structural properties of mixed micelles are unaffected by epigallocatechin gallate or Ile-Ile-Ala-Glu-Lys which integrate into the micellar surface; the diffusive motion of constituting lipids palmitoyl-oleoyl-phosphatidylcholine and cholesterol is significantly down-regulated by both epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys; CONCLUSIONS: The molecular mode of action of natural compounds epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys is a significant down-regulation of the diffusive motion of micellar lipids. GENERAL SIGNIFICANCE: Natural compounds like the green tea ingredient epigallocatechin gallate and a short pentapeptide, Ile-Ile-Ala-Glu-Lys, lead to a significant down-regulation of the diffusive motion of micellar lipids thereby modulating cholesterol absorption into physiological micelles.

Structural determinants in the bulk heterojunction.

Photovoltaics is one of the key areas in renewable energy research with remarkable progress made every year. Here we consider the case of a photoactive material and study its structural composition and the resulting consequences for the fundamental processes driving solar energy conversion. A multiscale approach is used to characterize essential molecular properties of the light-absorbing layer. A selection of bulk-representative pairs of donor/acceptor molecules is extracted from the molecular dynamics simulation of the bulk heterojunction and analyzed at increasing levels of detail. Significantly increased ground state energies together with an array of additional structural characteristics are identified that all point towards an auxiliary role of the material's structural organization in mediating charge-transfer and -separation. Mechanistic studies of the type presented here can provide important insights into fundamental principles governing solar energy conversion in next-generation photovoltaic devices.

Multiple Electronic and Structural Factors Control Cyclobutane Pyrimidine Dimer and 6-4 Thymine-Thymine Photodimerization in a DNA Duplex.

The T-T photodimerization paths leading to the formation of cyclobutane pyrimidine dimer (CPD) and 6-4 pyrimidine pyrimidone (64-PP), the two main DNA photolesions, have been resolved for a T-T step in a DNA duplex by two complementary state-of-the-art quantum mechanical approaches: QM(CASPT2//CASSCF)/MM and TD-DFT/PCM. Based on the analysis of several different representative structures, we define a new-ensemble of cooperating geometrical and electronic factors (besides the distance between the reacting bonds) ruling T-T photodimerization in DNA. CPD is formed by a barrierless path on an exciton state delocalized over the two bases. Large interbase stacking and shift values, together with a small pseudorotation phase angle for T at the 3'-end, favor this reaction. The oxetane intermediate, leading to a 64-PP adduct, is formed on a singlet T→T charge-transfer state and is favored by a large interbase angle and slide values. A small energy barrier (<0.3 eV) is associated to this path, likely contributing to the smaller quantum yield observed for this process. Eventually, a clear directionality is always shown by the electronic excitation characterizing the singlet photoactive state driving the photodimerization process: an exciton that is more localized on T3 and a 5'-T→3'-T charge transfer for CPD and oxetane formation, respectively, thus calling for specific electronic constraints.

Crossover of two power laws in the anomalous diffusion of a two lipid membrane.

Molecular dynamics simulations of a bi-layer membrane made by the same number of 1-palmitoyl-2-oleoyl-glycero-3-phospho-ethanolamine and palmitoyl-oleoyl phosphatidylserine lipids reveal sub-diffusional motion, which presents a crossover between two different power laws. Fractional Brownian motion is the stochastic mechanism that governs the motion in both regimes. The location of the crossover point is justified with simple geometrical arguments and is due to the activation of the mechanism of circumrotation of lipids about each other.

Excited state evolution of DNA stacked adenines resolved at the CASPT2//CASSCF/Amber level: from the bright to the excimer state and back.

Deactivation routes of bright ππ* (La) and excimer charge transfer (CT) states have been mapped for two stacked quantum mechanical (CASPT2//CASSCF) adenines inside a solvated DNA double strand decamer (poly(dA)·poly(dT)) described at the molecular mechanics level. Calculations show that one carbon (C2) puckering is a common relaxation coordinate for both the La and CT paths. By mapping the lowest crossing regions between La and CT states, together with the paths connecting the two states, we conclude that at least one CT state can be easily accessible. The lowest-lying conical intersections between ground state (GS) and CT states have been fully characterized in a realistic DNA environment for the first time. We show that the path to reach this crossing region from the CT minima involves high barriers that are not consistent with experimental data lifetimes. Instead, the multiexponential decay recorded in DNA, including the longest (ca. 100 picoseconds) lifetime component detected in oligomeric single- and double-stranded systems, is compatible with both intra-monomer relaxation processes along the La deactivation path (involving small barriers) and the population of the excimer (CT) state that behaves as a trap. In the latter case, deactivation is feasible only going back to the La state by following its preferred decay coordinate.

Common force field thermodynamics of cholesterol.

Four different force fields are examined for dynamic characteristics using cholesterol as a case study. The extent to which various types of internal degrees of freedom become thermodynamically relevant is evaluated by means of principal component analysis. More complex degrees of freedom (angle bending, dihedral rotations) show a trend towards force field independence. Moreover, charge assignments for membrane-embedded compounds are revealed to be critical with significant impact on biological reasoning.

GPU-accelerated computation of electron transfer.

Electron transfer is a fundamental process that can be studied with the help of computer simulation. The underlying quantum mechanical description renders the problem a computationally intensive application. In this study, we probe the graphics processing unit (GPU) for suitability to this type of problem. Time-critical components are identified via profiling of an existing implementation and several different variants are tested involving the GPU at increasing levels of abstraction. A publicly available library supporting basic linear algebra operations on the GPU turns out to accelerate the computation approximately 50-fold with minor dependence on actual problem size. The performance gain does not compromise numerical accuracy and is of significant value for practical purposes.

Local ice melting by an antifreeze protein.

Antifreeze proteins, AFP, impede freezing of bodily fluids and damaging of cellular tissues by low temperatures. Adsorption-inhibition mechanisms have been developed to explain their functioning. Using in silico Molecular Dynamics, we show that type I AFP can also induce melting of the local ice surface. Simulations of antifreeze-positive and antifreeze-negative mutants show a clear correlation between melting induction and antifreeze activity. The presence of local melting adds a function to type I AFPs that is unique to these proteins. It may also explain some apparently conflicting experimental results where binding to ice appears both quasipermanent and reversible.

Amyloid-ß fibril disruption by C60-molecular guidance for rational drug design.

The WHO has listed Alzheimer's disease among the major neurological disorders with an estimated 35 million people affected worldwide. Amyloid-ß is mostly believed to be the causative factor in Alzheimer's disease and the severity of the disease correlates with the tendency of amyloid-ß to form aggregation patterns-plaques. Lacking effective medication, the identification of any underlying mechanistic principles regarding plaque formation appears to be crucial. Here we carry out computer simulations to study the effect of C60 on structure and stability of an idealised pentameric construct of amyloid-ß units (a model fibril). A binding site on top of the structurally ordered stack of ß-sheets is identified that triggers structural alterations at the turn region of the hook-like ß-sheet assembly. Significant structural alterations are: (i) the destruction of regular helical twist, (ii) the loss of a stabilizing salt bridge and (iii) the loss of a stabilizing hydrophobic interaction close to the turn. Consequently, the main effect of C60 is the induction of sizable destabilization in native fibril structure. These structural insights may serve as a molecular guide for further rational drug design of effective inhibitors targeting fibril formation in Alzheimer's disease.

Structural features of aquaporin 4 supporting the formation of arrays and junctions in biomembranes.

A limited class of aquaporins has been described to form regular arrays and junctions in membranes. The biological significance of these structures, however, remains uncertain. Here we analyze the underlying physical principles with the help of a computational procedure that takes into account protein-protein as well as protein-membrane interactions. Experimentally observed array/junction structures are systematically (dis)assembled and major driving forces identified. Aquaporin 4 was found to be markedly different from the non-junction forming aquaporin 1. The environmental stabilization resulting from embedding into the biomembrane was identified as the main driving force. This highlights the role of protein-membrane interactions in aquaporin 4. Analysis of the type presented here can help to decipher the biological role of membrane arrays and junctions formed by aquaporin.

A computational analysis of the insertion of carbon nanotubes into cellular membranes.

Carbon nanotubes have been proposed to serve as nano-vehicles to deliver genetic or therapeutic material into the interior of cells because of their capacity to cross the cell membrane. A detailed picture of the molecular mode of action of such a delivery is, however, difficult to obtain because of the concealing effects of the cell membrane. Here we report a systematic computational study of membrane insertion of individual carbon nanotubes and carbon nanotube bundles using two entirely different and unrelated techniques. First a static scan of the environmental free energy is carried out based on a membrane mimicry approach and different insertion geometries are assessed. Then the dynamics is investigated with a coarse-grained approach that was previously used in the study of the integration dynamics of nanoparticles into the bilayer. The results of both models point, for unfunctionalized carbon nanotubes, at a preference for the horizontal orientation inside the internal hydrophobic layer of the cell membrane. Finally, the energetics of the formation of bundles of carbon nanotubes is studied. The cellular membrane promotes aggregation of carbon nanotubes in its hydrophobic core and modifies the structural stability of the bundles.

Theoretical mimicry of biomembranes.

The study of membrane proteins requires a proper consideration of the specific environment provided by the biomembrane. The compositional complexity of this environment poses great challenges to all experimental and theoretical approaches. In this article a rather simple theoretical concept is discussed for its ability to mimic the biomembrane. The biomembrane is approximated by three mimicry solvents forming individual continuum layers of characteristic physical properties. Several specific structural problems are studied with a focus on the biological significance of such an approach. Our results support the general perception that the biomembrane is crucial for correct positioning and embedding of its constituents. The described model provides a semi-quantitative tool of potential interest to many problems in structural membrane biology.

Current performance gains from utilizing the GPU or the ASIC MDGRAPE-3 within an enhanced Poisson Boltzmann approach.

Scientific applications do frequently suffer from limited compute performance. In this article, we investigate the suitability of specialized computer chips to overcome this limitation. An enhanced Poisson Boltzmann program is ported to the graphics processing unit and the application specific integrated circuit MDGRAPE-3 and resulting execution times are compared to the conventional performance obtained on a modern central processing unit. Speed Up factors are measured and an analysis of numerical accuracy is provided. On both specialized architectures the improvement is increasing with problem size and reaches up to a Speed Up factor of 39 x for the largest problem studied. This type of alternative high performance computing can significantly improve the performance of demanding scientific applications.

Dispersion terms and analysis of size- and charge dependence in an enhanced Poisson-Boltzmann approach.

We implement a well-established concept to consider dispersion effects within a Poisson-Boltzmann approach of continuum solvation of proteins. The theoretical framework is particularly suited for boundary element methods. Free parameters are determined by comparison to experimental data as well as high-level quantum mechanical reference calculations. The method is general and can be easily extended in several directions. The model is tested on various chemical substances and found to yield good-quality estimates of the solvation free energy without obvious indication of any introduced bias. Once optimized, the model is applied to a series of proteins, and factors such as protein size or partial charge assignments are studied.

Systematic study of the boundary composition in Poisson Boltzmann calculations.

We describe a three-stage procedure to analyze the dependence of Poisson Boltzmann calculations on the shape, size and geometry of the boundary between solute and solvent. Our study is carried out within the boundary element formalism, but our results are also of interest to finite difference techniques of Poisson Boltzmann calculations. At first, we identify the critical size of the geometrical elements for discretizing the boundary, and thus the necessary resolution required to establish numerical convergence. In the following two steps we perform reference calculations on a set of dipeptides in different conformations using the Polarizable Continuum Model and a high-level Density Functional as well as a high-quality basis set. Afterwards, we propose a mechanism for defining appropriate boundary geometries. Finally, we compare the classic Poisson Boltzmann description with the Quantum Chemical description, and aim at finding appropriate fitting parameters to get a close match to the reference data. Surprisingly, when using default AMBER partial charges and the rigorous geometric parameters derived in the initial two stages, no scaling of the partial charges is necessary and the best fit against the reference set is obtained automatically.

Parallel tempering molecular dynamics folding simulation of a signal peptide in explicit water.

Parallel temperature molecular dynamics simulations are used to explore the folding of a signal peptide, a short but functionally independent domain at the N-terminus of proteins. The peptide has been analyzed previously by NMR, and thus a solid reference state is provided with the experimental structure. Particular attention is paid to the role of water considered in full atomic detail. Different partial aspects in the folding process are quantified. The major group of obtained structures matches the NMR structure very closely. An important biological consequence is that in vivo folding of signal peptides seems to be possible within aqueous environments.

Beyond dimerization: a membrane-dependent activation model for interleukin-4 receptor-mediated signalling.

Class I cytokine receptors efficiently transfer activation signals from the extracellular space to the cytoplasm and play a dominant role in growth control and differentiation of human tissues. Although a significant body of literature is devoted to this topic, a consistent mechanistic picture for receptor activation in the membrane environment is still missing. Using the interleukin-4 receptor (IL-4R) as an example, we propose that the membrane-proximal stem-loop of the extracellular domains contains pivotal elements of a rotational switch. Interfacial energies of amino acid side-chains contained in the highly conserved WSXWS at the surface of the lipid bilayer suggest a new functional role for this motif. A generic activation mechanism for this receptor class is presented, which may impact the design of a new generation of biophysical assay systems.

Computational assessment of the entropy of solvation of small-sized hydrophobic entities.

A high level polarizable force field is used to study the temperature dependence of hydrophobic hydration of small-sized molecules from computer simulations. Molecular dynamics (MD) simulations of liquid water at various temperatures form the basis of free energy perturbation calculations that consider the onset and growth of a repulsive sphere. This repulsive sphere acts as a model construct for the hydrophobic species. In the present study, an extension is pursued for seven independent target temperatures, ranging from close to the freezing point almost up to the boiling point of liquid water under standard conditions. Care is taken to maintain proper physico-chemical model descriptions by cross-checking with experimental water densities at the selected target temperatures. The polarizable force field description of molecular water turns out to be suitable throughout the entire temperature domain considered. Derivatives of the computed free energies of hydrophobic hydration with respect to the temperature give access to the changes in entropy. In practice the entropy differential is determined from the negative of the slope of tangential lines formed at a certain target temperature in the free energy profile. The obtained changes in entropy are negative for small-sized cavities, and hence reconfirm the basic ideas of the Lum-Chandler-Weeks theory on hydrophobic hydration of small-sized solutes.

A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase.

CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting beta-strands 6 and 7. These residues are critical for CerK membrane association and polyphosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular mass as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast with most PH domains whose function strongly relies on charges located in the beta1-beta2 loop.