Kinetics of Huperzine A Dissociation from Acetylcholinesterase via Multiple Unbinding Pathways.

The dissociation of huperzine A (hupA) from Torpedo californica acetylcholinesterase ( TcAChE) was investigated by 4 µs unbiased and biased all-atom molecular dynamics (MD) simulations in explicit solvent. We performed our study using memetic sampling (MS) for the determination of reaction pathways (RPs), metadynamics to calculate free energy, and maximum-likelihood estimation (MLE) to recover kinetic rates from unbiased MD simulations. Our simulations suggest that the dissociation of hupA occurs mainly via two RPs: a front door along the axis of the active-site gorge (pwf) and through a new transient side door (pws), i.e., formed by the Ω-loop (residues 67-94 of TcAChE). An analysis of the inhibitor unbinding along the RPs suggests that pws is opened transiently after hupA and the Ω-loop reach a low free-energy transition state characterized by the orientation of the pyridone group of the inhibitor directed toward the Ω-loop plane. Unlike pws, pwf does not require large structural changes in TcAChE to be accessible. The estimated free energies and rates agree well with available experimental data. The dissociation rates along the unbinding pathways are similar, suggesting that the dissociation of hupA along pws is likely to be relevant. This indicates that perturbations to hupA- TcAChE interactions could potentially induce pathway hopping. In summary, our results characterize the slow-onset inhibition of TcAChE by hupA, which may provide the structural and energetic bases for the rational design of the next-generation slow-onset inhibitors with optimized pharmacokinetic properties for the treatment of Alzheimer's disease.

Charge separation and isolation in strong water droplet impacts.

Charge separation in condensed matter after strong impacts is a general and intriguing phenomenon in nature, which is often identified and described but not necessarily well understood in terms of a quantitative mechanistic picture. Here we show that charge separation naturally occurs if water droplets/clusters or ice particles with embedded charge carriers, e.g., ions, encounter a high energy impact with subsequent dispersion - even if the involved kinetic energy is significantly below the molecular ionization energy. We find that for low charge carrier concentrations (c < 0.01 mol L(-1)) a simple statistical Poisson model describes the charge distribution in the resulting molecular "fragments" or aggregates. At higher concentrations Coulomb interactions between the charge carriers become relevant, which we describe by a Monte Carlo approach. Our models are compared to experimental data for strong (laser) impacts on liquid micro beams and discussed for the charge generation in cluster-impact mass spectrometry on cosmic dust detectors where particle kinetic energies are below the plasma threshold. Taken together, a simple and intuitive but quantitative microscopic model is obtained, which may contribute to the understanding of a larger range of phenomena related to charge generation and separation in nature.

Hydrogen bond dynamics of superheated water and methanol by ultrafast IR-pump and EUV-photoelectron probe spectroscopy.

Supercritical water and methanol have recently drawn much attention in the field of green chemistry. It is crucial to an understanding of supercritical solvents to know their dynamics and to what extent hydrogen (H) bonds persist in these fluids. Here, we show that with femtosecond infrared (IR) laser pulses water and methanol can be heated to temperatures near and above their critical temperature Tc and their molecular dynamics can be studied via ultrafast photoelectron spectroscopy at liquid jet interfaces with high harmonics radiation. As opposed to previous studies, the main focus here is the comparison between the hydrogen bonded systems of methanol and water and their interpretation by theory. Superheated water initially forms a dense hot phase with spectral features resembling those of monomers in gas phase water. On longer timescales, this phase was found to build hot aggregates, whose size increases as a function of time. In contrast, methanol heated to temperatures near Tc initially forms a broad distribution of aggregate sizes and some gas. These experimental features are also found and analyzed in extended molecular dynamics simulations. Additionally, the simulations enabled us to relate the origin of the different behavior of these two hydrogen-bonded liquids to the nature of the intermolecular potentials. The combined experimental and theoretical approach delivers new insights into both superheated phases and may contribute to understand their different chemical reactivities.

Core hole screening and decay rates of double core ionized first row hydrides.

Because of the high intensity, X-ray free electron lasers allow one to create and probe double core ionized states in molecules. The decay of these multiple core ionized states crucially determines the evolution of radiation damage in single molecule diffractive imaging experiments. Here we have studied the Auger decay in hydrides of first row elements after single and double core ionization by quantum mechanical ab initio calculations. In our approach the continuum wave function of the emitted Auger electron is expanded into spherical harmonics on a radial grid. The obtained decay rates of double K-shell vacancies were found to be systematically larger than those for the respective single K-shell vacancies, markedly exceeding the expected factor of two. This enhancement is attributed to the screening effects induced by the core hole. We propose a simple model, which is able to predict core hole decay rates in molecules with low Z elements based on the electron density in the vicinity of the core hole.

Auger spectrum of a water molecule after single and double core ionization.

The high intensity of free electron lasers opens up the possibility to perform single-shot molecule scattering experiments. However, even for small molecules, radiation damage induced by absorption of high intense x-ray radiation is not yet fully understood. One of the striking effects which occurs under intense x-ray illumination is the creation of double core ionized molecules in considerable quantity. To provide insight into this process, we have studied the dynamics of water molecules in single and double core ionized states by means of electronic transition rate calculations and ab initio molecular dynamics (MD) simulations. From the MD trajectories, photoionization and Auger transition rates were computed based on electronic continuum wavefunctions obtained by explicit integration of the coupled radial Schrödinger equations. These rates served to solve the master equations for the populations of the relevant electronic states. To account for the nuclear dynamics during the core hole lifetime, the calculated electron emission spectra for different molecular geometries were incoherently accumulated according to the obtained time-dependent populations, thus neglecting possible interference effects between different decay pathways. We find that, in contrast to the single core ionized water molecule, the nuclear dynamics for the double core ionized water molecule during the core hole lifetime leaves a clear fingerprint in the resulting electron emission spectra. The lifetime of the double core ionized water was found to be significantly shorter than half of the single core hole lifetime.

Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1.

Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.

Progress in the analysis of membrane protein structure and function.

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at sub-nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.

Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF.

"Real time" molecular dynamics simulations of water permeation through human aquaporin-1 (AQP1) and the bacterial glycerol facilitator GlpF are presented. We obtained time-resolved, atomic-resolution models of the permeation mechanism across these highly selective membrane channels. Both proteins act as two-stage filters: Conserved fingerprint [asparagine-proline-alanine (NPA)] motifs form a selectivity-determining region; a second (aromatic/arginine) region is proposed to function as a proton filter. Hydrophobic regions near the NPA motifs are rate-limiting water barriers. In AQP1, a fine-tuned water dipole rotation during passage is essential for water selectivity. In GlpF, a glycerol-mediated "induced fit" gating motion is proposed to generate selectivity for glycerol over water.

A refined structure of human aquaporin-1.

A refined structure of the human water channel aquaporin-1 is presented. The model rests on the high resolution X-ray structure of the homologous bacterial glycerol transporter GlpF, electron crystallographic data at 3.8 A resolution and a multiple sequence alignment of the aquaporin superfamily. The crystallographic R and free R values (36.7% and 37.8%) for the refined structure are significantly lower than for previous models. Improved geometry and enhanced stability in molecular dynamics simulations demonstrate a significant improvement of the aquaporin-1 structure. Comparison with previous aquaporin-1 models shows significant differences, not only in the loop regions, but also in the core of the water channel.

Essential dynamics of reversible peptide folding: memory-free conformational dynamics governed by internal hydrogen bonds.

A principal component analysis has been applied on equilibrium simulations of a beta-heptapeptide that shows reversible folding in a methanol solution. The analysis shows that the configurational space contains only three dense sub-states. These states of relatively low free energy correspond to the "native" left-handed helix, a partly helical intermediate, and a hairpin-like structure. The collection of unfolded conformations form a relatively diffuse cloud with little substructure. Internal hydrogen-bonding energies were found to correlate well with the degree of folding. The native helical structure folds from the N terminus; the transition from the major folding intermediate to the native helical structure involves the formation of the two most C-terminal backbone hydrogen bonds. A four-state Markov model was found to describe transition frequencies between the conformational states within error limits, indicating that memory-effects are negligible beyond the nanosecond time-scale. The dominant native state fluctuations were found to be very similar to unfolding motions, suggesting that unfolding pathways can be inferred from fluctuations in the native state. The low-dimensional essential subspace, describing 69% of the collective atomic fluctuations, was found to converge at time-scales of the order of one nanosecond at all temperatures investigated, whereas folding/unfolding takes place at significantly longer time-scales, even above the melting temperature.

Molecular dynamics force probe simulations of antibody/antigen unbinding: entropic control and nonadditivity of unbinding forces.

Unbinding of a spin-labeled dinitrophenyl (DNP) hapten from the monoclonal antibody AN02 F(ab) fragment has been studied by force probe molecular dynamics (FPMD) simulations. In our nanosecond simulations, unbinding was enforced by pulling the hapten molecule out of the binding pocket. Detailed inspection of the FPMD trajectories revealed a large heterogeneity of enforced unbinding pathways and a correspondingly large flexibility of the binding pocket region, which exhibited induced fit motions. Principal component analyses were used to estimate the resulting entropic contribution of approximately 6 kcal/mol to the AN02/DNP-hapten bond. This large contribution may explain the surprisingly large effect on binding kinetics found for mutation sites that are not directly involved in binding. We propose that such "entropic control" optimizes the binding kinetics of antibodies. Additional FPMD simulations of two point mutants in the light chain, Y33F and I96K, provided further support for a large flexibility of the binding pocket. Unbinding forces were found to be unchanged for these two mutants. Structural analysis of the FPMD simulations suggests that, in contrast to free energies of unbinding, the effect of mutations on unbinding forces is generally nonadditive.

Exocytosis requires asymmetry in the central layer of the SNARE complex.

Assembly of SNAREs (soluble N:-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q-R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror R-->Q substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

Dynamic force spectroscopy of molecular adhesion bonds.

Recent advances in atomic force microscopy, biomembrane force probe experiments, and optical tweezers allow one to measure the response of single molecules to mechanical stress with high precision. Such experiments, due to limited spatial resolution, typically access only one single force value in a continuous force profile that characterizes the molecular response along a reaction coordinate. We develop a theory that allows one to reconstruct force profiles from force spectra obtained from measurements at varying loading rates, without requiring increased resolution. We show that spectra obtained from measurements with different spring constants contain complementary information.

The fold of human aquaporin 1.

The fold of human aquaporin 1 is determined from cryo-electron microscopic data at 4.5 A resolution. The monomeric structure consists of two transmembrane triple helices arranged around a pseudo-2-fold axis connected by a long flexible extracellular loop. Each triplet contains between its second and third helix a functional loop containing the highly conserved fingerprint NPA motif. These functional loops are assumed to fold inwards between the two triplets, thereby forming the heart of the water channel. The helix topology was determined from the directionality pattern of each of the six transmembrane helices with respect to the membrane, together with constraints defined by the sequence and atomic force microscopy data. The directionality of the helices was determined by collecting the best-fitting orientations resulting from a search through the three-dimensional experimental map for a large number of alpha-helical fragments. Tests on cryo-electron crystallographic bacteriorhodopsin data suggest that our method is generally applicable to determine the topology of helical proteins for which only medium-resolution electron microscopy data are available.

Ligand binding: molecular mechanics calculation of the streptavidin-biotin rupture force.

The force required to rupture the streptavidin-biotin complex was calculated here by computer simulations. The computed force agrees well with that obtained by recent single molecule atomic force microscope experiments. These simulations suggest a detailed multiple-pathway rupture mechanism involving five major unbinding steps. Binding forces and specificity are attributed to a hydrogen bond network between the biotin ligand and residues within the binding pocket of streptavidin. During rupture, additional water bridges substantially enhance the stability of the complex and even dominate the binding interactions. In contrast, steric restraints do not appear to contribute to the binding forces, although conformational motions were observed.

Self-organization of associative memory and pattern classification: recurrent signal processing on topological feature maps.

We extend the neural concepts of topological feature maps towards self-organization of auto-associative memory and hierarchical pattern classification. As is well-known, topological maps for statistical data sets store information on the associated probability densities. To extract that information we introduce a recurrent dynamics of signal processing. We show that the dynamics converts a topological map into an auto-associative memory for real-valued feature vectors which is capable to perform a cluster analysis. The neural network scheme thus developed represents a generalization of non-linear matrix-type associative memories. The results naturally lead to the concept of a feature atlas and an associated scheme of self-organized, hierarchical pattern classification.

Developmental changes in dichotic right ear advantage (REA).

A cross-sectional, dichotic listening study of 210 right-handed, middle-class children four to ten years old used thirty pairs of one-syllable words and thirty pairs of four-syllable numbers to assess the developmental course of ear asymmetry. A significant decrease in REA for both word and number pairs was found. Although right-ear and left-ear performance both increased with age, the developmental gain in left-ear performance was greater than the gain in right-ear performance, thus resulting in a decrease in REA with age. The results are discussed with respect to investigations which found no change in REA during development and a structural model based on the development of interhemispheric connectivity is proposed to explain the findings.