Dissociation Of Single Molecules Research Articles

Dynamics of Single-Molecule Dissociation by Selective Excitation of Molecular Phonons.

Breaking bonds selectively in molecules is vital in many chemistry reactions and custom nanoscale device fabrications. The scanning tunneling microscope (STM) has proved to be an ideal tool to initiate and view bond-selective chemistry at the single-molecule level, offering opportunities for the further study of the dynamics in single molecules on metal surfaces. We demonstrate H─HS and H─S bond breaking on Au(111) induced by tunneling electrons using low-temperature STM. An experimental study combined with theoretical calculations shows that the dissociation pathway is facilitated by vibrational excitations. Furthermore, the dissociation probabilities of the two different dissociation processes are bias dependent due to different inelastic-tunneling probabilities, and they are also closely linked to the lifetime of inelastic-tunneling electrons. Combined with time-dependent abinitio nonadiabatic molecular dynamics simulations, the dynamics of the injected electron and the phonon-excitation-induced molecule dissociation can be understood at the atomic scale, demonstrating the potential application of STM for the investigation of excited-state dynamics of single molecules on surfaces.

P53 Dynamically Directs TFIID Assembly on Target Gene Promoters.

p53 is a central regulator that turns on vast gene networks to maintain cellular integrity in the presence of various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key issue, we have undertaken an integrated approach involving single-molecule fluorescence microscopy, single-particle cryo-electron microscopy, and biochemistry. Our real-time single-molecule imaging data demonstrate that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID's ability to bind DNA by stabilizing TFIID contacts with both the core promoter and a region within p53's response element. Analysis of single-molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID's conversion to a rearranged DNA binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID's interaction with DNA induces p53 to rapidly dissociate, which likely leads to additional rounds of p53-mediated recruitment of other basal factors. Collectively, these findings indicate that p53 dynamically escorts and loads TFIID onto its target promoters.

A wireless centrifuge force microscope (CFM) enables multiplexed single-molecule experiments in a commercial centrifuge

The centrifuge force microscope (CFM) was recently introduced as a platform for massively parallel single-molecule manipulation and analysis. Here we developed a low-cost and self-contained CFM module that works directly within a commercial centrifuge, greatly improving accessibility and ease of use. Our instrument incorporates research grade video microscopy, a power source, a computer, and wireless transmission capability to simultaneously monitor many individually tethered microspheres. We validated the instrument by performing single-molecule force shearing of short DNA duplexes. For a 7 bp duplex, we observed over 1000 dissociation events due to force dependent shearing from 2 pN to 12 pN with dissociation times in the range of 10-100 s. We extended the measurement to a 10 bp duplex, applying a 12 pN force clamp and directly observing single-molecule dissociation over an 85 min experiment. Our new CFM module facilitates simple and inexpensive experiments that dramatically improve access to single-molecule analysis.

Ensemble and single-molecule biophysical characterization of D17.4 DNA aptamer–IgE interactions

BackgroundThe IgE-binding DNA aptamer 17.4 is known to inhibit the interaction of IgE with the high-affinity IgE Fc receptor FcεRI. While this and other aptamers have been widely used and studied, there has been relatively little investigation of the kinetics and energetics of their interactions with their targets, by either single-molecule or ensemble methods. MethodsThe dissociation kinetics of the D17.4/IgE complex and the effects of temperature and ionic strength were studied using fluorescence anisotropy and single-molecule spectroscopy, and activation parameters calculated. ResultsThe dissociation of D17.4/IgE complex showed a strong dependence on temperature and salt concentration. The koff of D17.4/IgE complex was calculated to be (2.92±0.18)×10−3s−1 at 50mM NaCl, and (1.44±0.02)×10−2s−1 at 300mM NaCl, both in 1mM MgCl2 and 25°C. The dissociation activation energy for the D17.4/IgE complex, Ea, was 16.0±1.9kcalmol−1 at 50mM NaCl and 1mM MgCl2. Interestingly, we found that the C19A mutant of D17.4 with stabilized stem structure showed slower dissociation kinetics compared to D17.4. Single-molecule observations of surface-immobilized D17.4/IgE showed much faster dissociation kinetics, and heterogeneity not observable by ensemble techniques. ConclusionsThe increasing koff value with increasing salt concentration is attributed to the electrostatic interactions between D17.4/IgE. We found that both the changes in activation enthalpy and activation entropy are insignificant with increasing NaCl concentration. The slower dissociation of the mutant C19A/IgE complex is likely due to the enhanced stability of the aptamer. General significanceThe activation parameters obtained by applying transition state analysis to kinetic data can provide details on mechanisms of molecular recognition and have applications in drug design. Single-molecule dissociation kinetics showed greater kinetic complexity than was observed in the ensemble in-solution systems, potentially reflecting conformational heterogeneity of the aptamer. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

Probing Single-Molecule Dissociations from a Bimolecular Complex NO-Co-Porphyrin.

Axial coordinations of diatomic NO molecules to metalloporphyrins play key roles in dynamic processes of biological functions such as blood pressure control and immune response. Probing such reactions at the single molecule level is essential to understand their physical mechanisms but has been rarely performed. Here we report on our single molecule dissociation experiments of diatomic NO from NO-Co-porphyrin complexes describing its dissociation mechanisms. Under tunneling junctions of scanning tunneling microscope, both positive and negative energy pulses gave rise to dissociations of NO with threshold voltages, +0.68 and -0.74 V at 0.1 nA tunneling current on Au(111). From the observed power law relations between dissociation rate and tunneling current, we argue that the dissociations were inelastically induced with molecular orbital resonances by stochastically tunneling electrons, which is supported with our density functional theory calculations. Our study shows that single molecule dissociation experiments can be used to probe reaction mechanisms in a variety of axial coordinations between small molecules and metalloporphyrins.

Ion Hydration Stripping Kinetics Support Thyrofluidic Ion Channel Gating

The thyrofluidic model of ion channel entry gating has the membrane electric field at the ion channel pore entrance responsible for stripping ion hydration shells, selectively admitting ions into the pore for transport. Capillary electrophoresis studies have shown that full hydration stripping occurs at field strengths corresponding to distances of 6.5 to 8 nanometers from a membrane. This work considers the rate at which hydration stripping occurs relative to the rate of ion migration near the membrane.The free energy barrier for one water molecule to escape the first (inner) hydration shell of a Na+ ion has been calculated to be 9.5 kJ/mole, equivalent to a field strength of 118.75 V/cm. Mean lifetime of attachment of one water molecule bound to a Na+ ion has been calculated to be 5 ps. A linear model of the energy and time to strip 5 of the 6 water molecules from the first shell yields 47.5 kJ/mol (594 V/cm) and 25 ps. Modeling single molecule dissociation to have exponential decay yields 101.7 kJ/mol (1,270 V/cm) and 197 ps. Electric field dependent ion velocity changes occur between 200 and 600 V/cm, consistent with the linear model.The Einstein-Smoluchowski equation shows ions with radius 0.1 nm diffuse at 3.2 nm/ns. As a result, Na+ will diffuse 0.08 nm (linear) or 0.63 nm (exponential) after reaching a field of 594 or 1,270 V/cm, respectively, for complete hydration stripping, the latter occurring 7 nm from the membrane. To date, all known kosmotropic ion channel pore entrances sit within 2 nm of the membrane. Thus, the membrane electric field will strip the hydration shells from Na+ long before the ion can reach the pore entrance. As such, electric field hydration stripping kinetics are compatible with thyrofluidic channel gating.

Dissociation Kinetics of an Enzyme−Inhibitor System Using Single-Molecule Force Measurements

We report on an improved method to interpret single molecule dissociation measurements using atomic force microscopy. We describe an easy to use methodology to reject nonspecific binding events, as well as estimating the number of multiple binding events. The method takes nonlinearities in the force profiles into account that result from the deformation of the used polymeric linkers. This new method is applied to a relevant enzyme-inhibitor system, latent matrix metalloprotease 9 (ProMMP-9, a gelatinase), and its inhibitor, tissue inhibitor of metalloproteases 1 (TIMP 1), which are important players in cancer metastasis. Our method provides a measured kinetic off-rate of 0.010 ± 0.003 s(-1) for the dissociation of ProMMP9 and TIMP1, which is consistent with values measured by ensemble methods.

Single-molecule force spectroscopy of supramolecular heterodimeric capsules

The specific interaction of a supramolecular binding motif was quantitatively evaluated by dynamic single-molecule force spectroscopy (SMFS) using an atomic force microscope (AFM). The supramolecular capsule forms by two different cavitands stitched together by four hydrogen bonds between carboxylic acid and pyridyl groups. The tetra(carboxyl)cavitand is monofunctionalized at the lower rim with a flexible poly(ethylene glycol) linker and attached to the AFM sensor tip. Single-molecule association experiments are accomplished using a diluted self-assembled monolayer (SAM) of the tetra(pyridyl)cavitand on a gold substrate. The measured single-molecule dissociation forces of the heterodimeric capsule represent the mechanical stability of the supramolecular system and allow a quantitative evaluation of the interaction according to the Bell-Evans model yielding dissociation rate constant k(off) = (0.14 +/- 0.14) s(-1), reaction length x(beta) = (0.56 +/- 0.076) nm and an estimated value of DeltaG(0) = -27 kJ mol(-1).

Association Kinetics from Single Molecule Force Spectroscopy Measurements

Single molecule force spectroscopy is often used to study the dissociation of single molecules by applying mechanical force to the intermolecular bond. These measurements provide the kinetic parameters of dissociation. We present what to our knowledge is a new atomic force microscopy-based approach to obtain the activation energy of the association reaction and approximate grafting density of reactive receptors using the dependence of the probability to form molecular bonds on probe velocity when one of the interacting molecules is tethered by a flexible polymeric linker to the atomic force microscopy probe. Possible errors in the activation energy measured with this approach are considered and resulting corrections are included in the data analysis. This new approach uses the same experimental setup as traditional force spectroscopy measurements that quantify dissociation kinetics. We apply the developed methodology to measure the activation energy of biotin-streptavidin association (including a contribution from the steric factor) and obtain a value of 8 ± 1 kT. This value is consistent with the association rate measured previously in solution. Comparison with the solution-derived activation energy indicates that kinetics of biotin-streptavidin binding is mainly controlled by the reaction step.

Orbital specific chemistry: Controlling the pathway in single-molecule dissociation

A scanning tunneling microscope (STM) was used to control the pathway of the dissociation of single O(2) molecules chemisorbed on Ag(110) at 13 K. Tunneling of electrons from the STM tip into the O(2) caused dissociation of the molecule, giving rise to two adsorbed O atoms separated along the [110] direction. In contrast, the ejection of electrons from the O(2) molecule produced adsorbed O atoms separated along the [001] direction. These results illustrate that control of the dissociation pathway and product formation are associated with a specific molecular orbital located at the Fermi level.

Two-electron dissociation of single molecules by atomic manipulation at room temperature

Using the tip of a scanning tunnelling microscope (STM) to mechanically manipulate individual atoms and molecules on a surface is now a well established procedure. Similarly, selective vibrational excitation of adsorbed molecules with an STM tip to induce motion or dissociation has been widely demonstrated. Such experiments are usually performed on weakly bound atoms that need to be stabilized by operating at cryogenic temperatures. Analogous experiments at room temperature are more difficult, because they require relatively strongly bound species that are not perturbed by random thermal fluctuations. But manipulation can still be achieved through electronic excitation of the atom or molecule by the electron current tunnelling between STM tip and surface at relatively high bias voltages, typically 1-5 V. Here we use this approach to selectively dissociate chlorine atoms from individual oriented chlorobenzene molecules adsorbed on a Si(111)-7 x 7 surface. We map out the final destination of the chlorine daughter atoms, finding that their radial and angular distributions depend on the tunnelling current and hence excitation rate. In our system, one tunnelling electron has nominally sufficient energy to induce dissociation, yet the process requires two electrons. We explain these observations by a two-electron mechanism that couples vibrational excitation and dissociative electron attachment steps.

Comparison of experimental estimates and model predictions of protein crystal nucleation rates

Estimates of protein crystal nucleation rates determined from three independent sets of experimental observations are compared for internal consistency. Large discrepancies emerge. A qualitative understanding of these discrepancies is gained from an analysis of the experimental techniques used within the framework of extant models for nucleation rates. Quantitatively, however, these models fail to predict nucleation rates of protein crystals. Possible origins of this failure lie in the approximate descriptions of the interaction potentials between protein molecules and the processes of aggregation and dissociation of single molecules on cluster surfaces that lead to crystal nucleation.

Physical and Biological Properties of Cationic Triesters of Phosphatidylcholine

The properties of a new class of phospholipids, alkyl phosphocholine triesters, are described. These compounds were prepared from phosphatidylcholines through substitution of the phosphate oxygen by reaction with alkyl trifluoromethylsulfonates. Their unusual behavior is ascribed to their net positive charge and absence of intermolecular hydrogen bonding. The O-ethyl, unsaturated derivatives hydrated to generate large, unilamellar liposomes. The phase transition temperature of the saturated derivatives is very similar to that of the precursor phosphatidylcholine and quite insensitive to ionic strength. The dissociation of single molecules from bilayers is unusually facile, as revealed by the surface activity of aqueous liposome dispersions. Vesicles of cationic phospholipids fused with vesicles of anionic lipids. Liquid crystalline cationic phospholipids such as 1,2-dioleoyl- sn-glycero-3-ethylphosphocholine triflate formed normal lipid bilayers in aqueous phases that interacted with short, linear DNA and supercoiled plasmid DNA to form a sandwich-structured complex in which bilayers were separated by strands of DNA. DNA in a 1:1 (mol) complex with cationic lipid was shielded from the aqueous phase, but was released by neutralizing the cationic charge with anionic lipid. DNA-lipid complexes transfected DNA into cells very effectively. Transfection efficiency depended upon the form of the lipid dispersion used to generate DNA-lipid complexes; in the case of the O-ethyl derivative described here, large vesicle preparations in the liquid crystalline phase were most effective.

Inducing and imaging single molecule dissociation on a semiconductor surface: H2S and D2S on Si(111)-7×7

Using a variable-temperature, ultrahigh vacuum scanning tunneling microscope (STM), we have induced and imaged the dissociation of H2S and D2S on Si(111)-7×7. H2S and D2S adsorb dissociatively at low coverage, from 50 to 300 K. Individual HS (or DS) fragments can be further dissociated with the STM at low temperatures without affecting neighboring adsorbates. The hydrogen (deuterium) atom either desorbs or re-attaches to a nearby silicon atom. Near room temperature (297 K) and above, DS dissociates thermally, with an activation barrier of 0.73±0.15 eV. The activation barrier was calculated from atomistic studies of the dissociation rates at temperatures between 297 and 312 K.