Dissociation Events Research Articles

Distinct Dopamine D₂ Receptor Antagonists Differentially Impact D₂ Receptor Oligomerization.

Dopamine D2 receptors (D2R) are known to form transient homodimer complexes, of which the increased formation has already been associated with development of schizophrenia. Pharmacological targeting and modulation of the equilibrium of these receptor homodimers might lead to a better understanding of the critical role played by these complexes in physiological and pathological conditions. Whereas agonist addition has shown to prolong the D2R dimer lifetime and increase the level of dimer formation, the possible influence of D2R antagonists on dimerization has remained rather unexplored. Here, using a live-cell reporter assay based on the functional complementation of a split Nanoluciferase, a panel of six D2R antagonists were screened for their ability to modulate the level of D2LR dimer formation. Incubation with the D2R antagonist spiperone decreased the level of D2LR dimer formation significantly by 40–60% in real-time and after long-term (≥16 h) incubations. The fact that dimer formation of the well-studied A2a–D2LR dimer was not altered following incubation with spiperone supports the specificity of this observation. Other D2R antagonists, such as clozapine, risperidone, and droperidol did not significantly evoke this dissociation event. Furthermore, molecular modeling reveals that spiperone presents specific Tyr1995.48 and Phe3906.52 conformations, compared to clozapine, which may determine D2R homodimerization.

Investigating the stability of the SecA–SecYEG complex during protein translocation across the bacterial membrane

During posttranslational translocation in Escherichia coli, polypeptide substrates are driven across the membrane through the SecYEG protein-conducting channel using the ATPase SecA, which binds to SecYEG and couples nucleotide hydrolysis to polypeptide movement. Recent studies suggest that SecA is a highly dynamic enzyme, able to repeatedly bind and dissociate from SecYEG during substrate translocation, but other studies indicate that these dynamics, here referred to as "SecA processivity," are not a requirement for transport. We employ a SecA mutant (PrlD23) that associates more tightly to membranes than WT SecA, in addition to a SecA-SecYEG cross-linked complex, to demonstrate that SecA-SecYEG binding and dissociation events are important for efficient transport of the periplasmic protein proPhoA. Strikingly however, we find that transport of the precursor of the outer membrane protein proOmpA does not depend on SecA processivity. By exchanging signal sequence and protein domains of similar size between PhoA and OmpA, we find that SecA processivity is not influenced by the sequence of the protein substrate. In contrast, using an extended proOmpA variant and a truncated derivative of proPhoA, we show that SecA processivity is affected by substrate length. These findings underscore the importance of the dynamic nature of SecA-SecYEG interactions as a function of the preprotein substrate, features that have not yet been reported using other biophysical or in vivo methods.

Illuminating the Black Box of DNA-Protein Interactions

The biological functions of DNA rely not just on its raw nucleotide sequence but also on a suite of proteins which interact with DNA. Many of these interactions have a significant influence on the topology of DNA and its subsequent role inside living cells: several basic, essential processes such as DNA replication, transcription and gene regulation are all dependent in highly sensitive ways upon these often complex and heterogeneous binding and dissociation events. Here I will discuss some of our insights in this area, which have all stemmed from the development and application of in vitro, in silico and in vivo methods, that could, perhaps, be thought of as emerging from the growing toolkit of ‘next generation biophysics’. This powerful combinatorial approach has enabled us to shed some light into the black box of DNA-protein interactions at the level of single, functional molecules.

An Experimental Investigation of Gas-Production Rates During Depressurization of Sedimentary Methane Hydrates

Summary Sedimentary methane hydrates contain a vast amount of untapped natural gas that can be produced through pressure depletion. Several field pilots have proved the concept with days to weeks of operation, but the longer-term response remains uncertain. This paper investigates the parameters affecting the rate of gas recovery from methane-hydrate-bearing sediments. The recovery of methane gas from hydrate dissociation through pressure depletion was studied at different initial hydrate saturations and different constant production pressures in cylindrical sandstone cores. Core-scale dissociation patterns were mapped with magnetic resonance imaging (MRI), and pore-scale dissociation events were visualized in a high-pressure micromodel. Key findings from the gas-production-rate analysis are that the maximum rate of recovery is only to a small extent affected by the magnitude of the pressure reduction below the dissociation pressure, and that the hydrate saturation directly affects the rate of recovery, where intermediate hydrate saturations (0.30 to 0.50) give the highest initial recovery rate. These results are of interest to anyone who evaluates the production performance of sedimentary hydrate accumulations and demonstrate how important accurate saturation estimates are to prediction of both the initial rate of gas recovery and the ultimate-recovery efficiency.

High Resolution Melting Analysis (HRMA) and its strategic applications in Veterinary science

High Resolution Melting Analysis (HRMA) is a simple, PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex. To follow the transition of double-stranded DNA (dsDNA) to singlestranded (ssDNA), intercalating saturating dyes such as LC Green, EvaGreen, Chromofy and SYTO 9 were employed. HRM is based on the analysis of fluorescence curves produced by DNA intercalating dye during strand dissociation events in the melting phase following real-time PCR. The melting temperature at which 50% of the DNA is in the double stranded state can be approximated by taking the derivative of the melting curve. The distinctive melting curve can be used to detect DNA sequence variants without the need for any post-PCR handling. It is a rapid, cost effective, less complicated method with high sensitivity and specificity. The strategic use of this methodology includes identification of Single Nucleotide Polymorphism (SNPs), genotyping, species identification and detection of genetic variants related to antimicrobial resistance. The simplicity, low cost and high efficiency of the technique is promising to take leading role in biological research in veterinary science in future.

Single-Molecule Kinetic Fingerprinting for the Ultrasensitive Detection of Small Molecules with Aptasensors.

Aptamers have emerged as promising molecular tools for small-molecule analyte sensing. However, the performance of such aptasensors is generally limited by leakage since it has been difficult to completely suppress signal in the absence of analyte, resulting in a compromise between sensitivity and specificity. Here, we describe a methodology for the ultrasensitive detection of analytes combining aptasensors with single-molecule kinetic fingerprinting. A short, fluorescently labeled DNA probe is utilized to detect the structural changes upon ligand binding to the designed hairpin-shaped aptasensor probe. The Poisson statistics of binding and dissociation events of the DNA probe to single surface-immobilized aptasensor molecules is monitored by total internal reflection fluorescence microscopy, permitting the high-accuracy discrimination of the ligand bound and ligand-free states, resulting in zero background. The programmable dynamics of the hairpin enables fine-tuning of the hybridization kinetics of the fluorescent probe, rendering the acquisition time sufficiently flexible to optimize discrimination. Remarkable detection limits are achieved for a diverse set of analytes when spiked into chicken meat extract: the nucleotide adenosine (0.3 pM), the insecticide acetamiprid (0.35 pM), and the dioxin-like toxin PCB-77 (0.72 pM), which is superior to recently reported aptasensors. Our generalizable method significantly improves the performance of aptasensors, with the potential to extend to other molecular biomarkers.

Kinetics of Ligand-Protein Dissociation from All-Atom Simulations: Are We There Yet?

Large parallel gains in the development of both computational resources and sampling methods have now made it possible to simulate dissociation events in ligand-protein complexes with all-atom resolution. Such encouraging progress, together with the inherent spatiotemporal resolution associated with molecular simulations, has left their use for investigating dissociation processes brimming with potential, both in rational drug design, where it can be an invaluable tool for determining the mechanistic driving forces behind dissociation rate constants, and in force-field development, where it can provide a catalog of transient molecular structures with which to refine force fields. Although much progress has been made in making force fields more accurate, reducing their error for transient structures along a transition path could yet prove to be a critical development helping to make kinetic predictions much more accurate. In what follows, we will provide a state-of-the-art compilation of the enhanced sampling methods based on molecular dynamics (MD) simulations used to investigate the kinetics and mechanisms of ligand-protein dissociation processes. Due to the time scales of such processes being slower than what is accessible using straightforward MD simulations, several ingenious schemes are being devised at a rapid rate to overcome this obstacle. Here we provide an up-to-date compendium of such methods and their achievements and shortcomings in extracting mechanistic insight into ligand-protein dissociation. We conclude with a critical and provocative appraisal attempting to answer the title of this Perspective.

Toward Achieving Efficient and Accurate Ligand-Protein Unbinding with Deep Learning and Molecular Dynamics through RAVE.

In this work, we demonstrate how to leverage our recent iterative deep learning-all atom molecular dynamics (MD) technique "Reweighted autoencoded variational Bayes for enhanced sampling (RAVE)" (Ribeiro, Bravo, Wang, Tiwary, J. Chem. Phys. 2018, 149, 072301) for investigating ligand-protein unbinding mechanisms and calculating absolute binding free energies, Δ Gb, when plagued with difficult to sample rare events. In order to do so, we introduce a simple but powerful extension to RAVE that allows learning a reaction coordinate expressed as a piecewise function that is linear over all intervals. Such an approach allows us to retain the physical interpretation of a RAVE-derived reaction coordinate while making the method more applicable to a wider range of complex biophysical problems. As we will demonstrate, using as our test-case the slow dissociation of benzene from the L99A variant of lysozyme, the RAVE extension led to observing an unbinding event in 100% of the independent all-atom MD simulations, all within 3-50 ns for a process that takes on an average close to few hundred milliseconds, which reflects a 7 orders of magnitude acceleration relative to straightforward MD. Furthermore, we will show that without the use of time-dependent biasing, clear back-and-forth movement between metastable intermediates was achieved during the various simulations, demonstrating the caliber of the RAVE-derived piecewise reaction coordinate and bias potential, which together drive efficient and accurate sampling of the ligand-protein dissociation event. Last, we report the results for Δ Gb, which via very short MD simulations, can form a strict lower-bound that is ∼2-3 kcal/mol off from experiments. We believe that RAVE, together with its multidimensional extension that we introduce here, will be a useful tool for simulating the slow unbinding process of practical ligand-protein complexes in an automated manner with minimal use of human intuition.

Crystal Plasticity Modeling of Anisotropic Hardening and Texture Due to Dislocation Transmutation in Twinning.

In crystalline materials, dislocations are three-dimensional lattice distortions that systematically distort twin interfaces that they encounter. This results in dislocation dissociation events and changes in the atomic structure of the interface. The manner in which the interface distorts drive the product of the dissociation event, and consequently, the incident dislocation core and the magnitude and relative direction of the Burgers vector govern these slip-twin interaction phenomena. Recent characterization studies using transmission electron microscopy as well as advanced molecular dynamic simulations have shown that slip dislocations, whether striking or struck by a {102} twin boundary, dissociate into a combination of twinning disconnections, interfacial disclinations (facets), jogs, and other types of dislocations engulfed inside the twin domains, called transmuted dislocations. While twinning disconnections were found to promote twin propagation, the dislocations incorporated inside the twin are of considerable importance to hardening and damage initiation as they more significantly obstruct slip dislocations accommodating plasticity of the twins. In this work, the dislocation transmutation event and its effect on hardening is captured using a dislocation density based hardening model contained in a visco-plastic self-consistent mean-field model. This is done by allowing the twins to increase their dislocation densities, not only by virtue of slip inside the twin, but also through dislocations that transmute from the parents as the twin volume fraction increases. A correspondence matrix rule is used to determine the type of converted dislocations while tracking and parameterizing their evolution. This hypothesis provides a modeling framework for capturing slip-twin interactions. The model is used to simulate the mechanical response of pure Mg and provides a more physically based approach for modeling stress-strain behavior.

Detection of Neutral CO Lost During Ionic Dissociation Using Atmospheric Pressure Thermal Dissociation Mass Spectrometry (APTD-MS).

Elucidation of ion dissociation patterns is particularly important to structural analysis by mass spectrometry (MS). However, typically, only the charged fragments from an ion dissociation event are detected in tandem MS experiments; neutrals are not identified. In recent years, we have developed an atmospheric pressure thermal dissociation (APTD) technique that can be applied to dissociate ions at atmosphere pressure and thus provide one way to characterize neutral fragments. In this paper, we focus on the detection of neutral CO resulting from amino acid and peptide ion dissociation. In the first set of experiments, several protonated amino acids (e.g., + 1 ion of phenylalanine) were found to undergo loss of a neutral (s) of total mass 46Da, a process leading to iminium ion formation. We successfully detected the neutral species CO by using a CO sensor, UV-Vis and MS analysis following selective CO trapping with a rhodium complex. The capture of CO from dissociation of protonated amino acids supports the assignment of the loss of 46Da to neutral losses of CO and H2O, rather than loss of formaldehyde or dihydroxycarbene, other possible fragmentation pathways that have been subject of debate for a long time. In a second experiment, we used the APTD method in combination with the CO detection technique, to demonstrate the formation of CO in the conversion of b ions to a ions during peptide ion dissociations. These results showed the potential of APTD in the elucidation of ion dissociation mechanisms, using simple home-built apparatus. Graphical Abstract ᅟ.

Measurement of charged particle spectra in minimum-bias events from proton\u2013proton collisions at sqrt{s}=13,text {TeV}

Pseudorapidity, transverse momentum, and multiplicity distributions are measured in the pseudorapidity range |eta | < 2.4 for charged particles with transverse momenta satisfying p_{mathrm {T}} > 0.5,text {GeV} in proton–proton collisions at a center-of-mass energy of sqrt{s} = 13,text {TeV} . Measurements are presented in three different event categories. The most inclusive of the categories corresponds to an inelastic mathrm {p}mathrm {p} data set, while the other two categories are exclusive subsets of the inelastic sample that are either enhanced or depleted in single diffractive dissociation events. The measurements are compared to predictions from Monte Carlo event generators used to describe high-energy hadronic interactions in collider and cosmic-ray physics.

Rapidity gap distribution in diffractive deep-inelastic scattering and parton genealogy

We propose a partonic picture for high-mass diffractive dissociation events in onium-nucleus scattering, which leads to simple and robust predictions for the distribution of the sizes of gaps in diffractive dissociation of virtual photons off nuclei at very high energies. We show that the obtained probability distribution can formally be identified to the distribution of the decay time of the most recent common ancestor of a set of objects generated near the edge of a branching random walk, and explain the physical origin of this appealing correspondence. We then use the fact that the diffractive cross section conditioned to a minimum rapidity gap size obeys a set of Balitsky-Kovchegov equations in order to test numerically our analytical predictions. Furthermore, we show how simulations in the framework of a Monte Carlo implementation of the QCD evolution support our picture.

Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices

We describe a tethered multifluorophore motion assay based on DNA origami for revealing bimolecular reaction kinetics on the single-molecule level. Molecular binding partners may be placed at user-defined positions and in user-defined stoichiometry; and binding states are read out by tracking the motion of quickly diffusing fluorescent reporter units. Multiple dyes per reporter unit enable singe-particle observation for more than 1 hour. We applied the system to study in equilibrium reversible hybridization and dissociation of complementary DNA single strands as a function of tether length, cation concentration, and sequence. We observed up to hundreds of hybridization and dissociation events per single reactant pair and could produce cumulative statistics with tens of thousands of binding and unbinding events. Because the binding partners per particle do not exchange, we could also detect subtle heterogeneity from molecule to molecule, which enabled separating data reflecting the actual target strand pair binding kinetics from falsifying influences stemming from chemically truncated oligonucleotides. Our data reflected that mainly DNA strand hybridization, but not strand dissociation, is affected by cation concentration, in agreement with previous results from different assays. We studied 8-bp-long DNA duplexes with virtually identical thermodynamic stability, but different sequences, and observed strongly differing hybridization kinetics. Complementary full-atom molecular-dynamics simulations indicated two opposing sequence-dependent phenomena: helical templating in purine-rich single strands and secondary structures. These two effects can increase or decrease, respectively, the fraction of strand collisions leading to successful nucleation events for duplex formation.

Conformational sampling of membranes by Akt controls its activation and inactivation

The protein kinase Akt controls myriad signaling processes in cells, ranging from growth and proliferation to differentiation and metabolism. Akt is activated by a combination of binding to the lipid second messenger PI(3,4,5)P3 and its subsequent phosphorylation by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2. The relative contributions of these mechanisms to Akt activity and signaling have hitherto not been understood. Here, we show that phosphorylation and activation by membrane binding are mutually interdependent. Moreover, the converse is also true: Akt is more rapidly dephosphorylated in the absence of PIP3, an autoinhibitory process driven by the interaction of its PH and kinase domains. We present biophysical evidence for the conformational changes in Akt that accompany its activation on membranes, show that Akt is robustly autoinhibited in the absence of PIP3 irrespective of its phosphorylation, and map the autoinhibitory PH-kinase interface. Finally, we present a model for the activation and inactivation of Akt by an ordered series of membrane binding, phosphorylation, dissociation, and dephosphorylation events.

Human RAD51 rapidly forms intrinsically dynamic nucleoprotein filaments modulated by nucleotide binding state.

Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an ‘open’ conformation when compared to a ‘closed’ structure formed by mutants, reflecting alterations in helical pitch. The kinetics of formation/disassembly of RAD51 filaments show rapid and high ssDNA coverage via low cooperativity binding of RAD51 units along the DNA. Subsequently, a series of isomerization or dissociation events mediated by nucleotide binding state creates intrinsically dynamic RAD51 NFs. Our findings highlight important a mechanistic divergence among recombinases from different organisms, in line with the diversity of biological mechanisms of HR initiation and quality control. These data reveal unexpected intrinsic dynamic properties of the RAD51 filament during assembly/disassembly, which may be important for the proper control of homologous recombination.

Photodissociation dynamics of the simplest alkyl peroxy radicals, CH3OO and C2H5OO, at 248 nm.

The photodissociation dynamics of the simplest alkyl peroxy radicals, methyl peroxy (CH3OO) and ethyl peroxy (C2H5OO), are investigated using fast beam photofragment translational spectroscopy. A fast beam of CH3OO- or C2H5OO- anions is photodetached to generate neutral radicals that are subsequently dissociated using 248 nm photons. The coincident detection of the photofragment positions and arrival times allows for the determination of mass, translational energy, and angular distributions for both two-body and three-body dissociation events. CH3OO exhibits repulsive O loss resulting in the formation of O(1D) + CH3O with high translational energy release. Minor two-body channels leading to OH + CH2O and CH3O + O(3P) formation are also detected. In addition, small amounts of H + O(3P) + CH2O are observed and attributed to O loss followed by CH3O dissociation. C2H5OO exhibits more complex dissociation dynamics, in which O loss and OH loss occur in roughly equivalent amounts with O(1D) formed as the dominant O atom electronic state via dissociation on a repulsive surface. Minor two-body channels leading to the formation of O2 + C2H5 and HO2 + C2H4 are also observed and attributed to a ground state dissociation pathway following internal conversion. Additionally, C2H5OO dissociation yields a three-body product channel, CH3 + O(3P) + CH2O, for which the proposed mechanism is repulsive O loss followed by the dissociation of C2H5O over a barrier. These results are compared to a recent study of tert-butyl peroxy (t-BuOO) in which 248 nm excitation results in three-body dissociation and ground state two-body dissociation but no O(1D) production.

Visualization of Functional Structure and Kinetic Dynamics of Cellulases.

Cellulose is the most abundant carbohydrate on earth and hydrolyzed by cellulases in nature. During catalysis, cellulase transfers protons to and from the oxygen atoms of the glycosidic bond and a water molecule. Since cellulose is an insoluble polymer, some kinds of cellulases, with high activity toward crystalline cellulose, move on the crystal surface with continuous hydrolysis of the molecular chain. In addition, binding and dissociation on/from the crystal surface are also important elementary steps of the reaction cycle. Recently, these interesting features of cellulases can be directly analyzed, due to the development of visualization techniques. In this chapter, we introduce (1) visualization of the protonation state of the catalytic residue by neutron crystallography, (2) visualization of processive movement on the crystal surface by high-speed atomic force microscopy , and (3) visualization of binding and dissociation events by single-molecule fluorescence microscopy.

Product-oriented chemical surface modification of a levansucrase (SacB) via an ene-type reaction.

Carbohydrate processing enzymes are sophisticated tools of living systems that have evolved to execute specific reactions on sugars. Here we present for the first time the site-selective chemical modification of exposed tyrosine residues in SacB, a levansucrase from Bacillus megaterium (Bm-LS) for enzyme engineering purposes via an ene-type reaction. Bm-LS is unable to sustain the synthesis of high molecular weight (HMW) levan (a fructose polymer) due to protein-oligosaccharide dissociation events occurring at an early stage during polymer elongation. We switched the catalyst from levan-like oligosaccharide synthesis to the efficient production of a HMW fructan polymer through the covalent addition of a flexible chemical side-chain that fluctuates over the central binding cavity of the enzyme preventing premature oligosaccharide disengagement.

Decoding the linear viscoelastic properties of model telechelic metallo-supramolecular polymers

This work focuses on the linear viscoelastic properties of entangled telechelic bulk metallo-supramolecular polymers. The latter are based on linear and star poly(n-butyl acrylate)s functionalized with a terpyridine ligand at each chain extremity, in the presence of transition metal ion of varying nature. The systems are investigated both experimentally and theoretically using small amplitude oscillatory shear and a modified version of the tube-based time marching algorithm (TMA), respectively. The experimental data reveal that sample relaxation depends on both disentanglement and association dynamics, with the respective importance of these two processes depending on the nature of the metal ion and on the temperature. A good description of the data is achieved using the modified TMA model, provided that dissociation events of metal-ligand complexes occur via ligand exchange. The model contains two fitting parameters, i.e., the fraction of unassociated stickers and the longest time needed in order to ensu...

Investigation of the two- and three-fragment photodissociation of the tert-butyl peroxy radical at 248 nm.

The photodissociation dynamics of the tert-butyl peroxy (t-BuOO) radical are studied by fast-radical-beam coincidence translational spectroscopy. The neutral t-BuOO radical is formed by photodetachment of the corresponding t-BuOO- anion at 700 nm (1.77 eV), followed by dissociation at 248 nm (5.00 eV). Photofragment mass and translational energy distributions are obtained. The major channel is found to be three-body fragmentation to form O, CH3, and acetone (83%), with minor two-body fragmentation channels leading to the formation of O2 + tert-butyl radical (10%) and HO2 + isobutene (7%). Experimental results show that the translational energy distribution for two-body dissociation peaks is close to zero translational energy, with an isotropic angular distribution of fragments. These results indicate that two-body fragmentation proceeds via internal conversion to the ground electronic state followed by statistical dissociation. For three-body dissociation, the translational energy distribution peaks closer to the maximal allowed translational energy and shows an anisotropic distribution of the plane of the dissociating fragments, implying rapid dissociation on an excited-state surface. A small shoulder in the three-body translational energy distribution suggests that some three-fragment dissociation events proceed by a different mechanism, involving internal conversion to the ground electronic state followed by sequential dissociation.