Intermittent single-molecule interfacial electron transfer dynamics

We report on single-molecule studies of photosensitized interfacial electron transfer (ET) processes in Coumarin 343 (C343)-TiO(2) nanoparticles (NP) and Cresyl Violet (CV(+))-TiO(2) NP systems, using time-correlated single-photon counting coupled with scanning confocal fluorescence microscopy. Fluorescence intensity trajectories of individual dye molecules adsorbed on a semiconductor NP surface showed fluorescence fluctuations and blinking, with time constants distributed from milliseconds to seconds. The fluorescence fluctuation dynamics were found to be inhomogeneous from molecule to molecule and from time to time, showing significant static and dynamic disorders in the interfacial ET reaction dynamics. We attribute fluorescence fluctuations to the interfacial ET reaction rate fluctuations, associating redox reactivity intermittency with the fluctuations of molecule-TiO(2) electronic and Franck-Condon coupling. Intermittent interfacial ET dynamics of individual molecules could be characteristic of a surface chemical reaction strongly involved with and regulated by molecule-surface interactions. The intermittent interfacial reaction dynamics that likely occur among single molecules in other interfacial and surface chemical processes can typically be observed by single-molecule studies but not by conventional ensemble-averaged experiments.     

Identifying the mechanisms of polymer friction through molecular dynamics simulation

Mechanisms governing the tribological behavior of polymer-on-polymer sliding were investigated by molecular dynamics simulations. Three main mechanisms governing frictional behavior were identified. Interfacial "brushing" of molecular chain ends over one another was observed as the key contribution to frictional forces. With an increase of the sliding speed, fluctuations in frictional forces reduced in both magnitude and periodicity, leading to dynamic frictional behavior. While "brushing" remained prevalent, two additional irreversible mechanisms, "combing" and "chain scission", of molecular chains were observed when the interfaces were significantly diffused.     

Analysis of single-molecule dynamics in liquid HF

The center-of-mass velocity autocorrelation function of liquid HF is examined by means of a velocity field approach, originally developed for monatomic liquids and subsequently tested for liquid water. The basic ingredients of the approach (the wavevector-dependent current correlations) are evaluated by computer simulations in a model HF system using a recently developed intermolecular potential. The theory is found to reproduce the relevant features of self-dynamics in HF. The main advantage of the approach is the decomposition of single-molecule motion into longitudinal and transverse contributions, which provides information clarifying the origin of the peculiarities of HF dynamics. A comparison with the results for H₂O is presented, with the aim of stressing the different behavior of these two hydrogen-bonded systems.     

An important step towards single-molecule reaction dynamics

正The detection of chemical reaction dynamics at the singlemolecule level is of crucial importance for both scientific studies and industrial applications.As the intrinsic mechanism of a chemical reaction can be uncovered during such a unique detection,it offers an opportunity to understand and control reactions in pharmaceutical engineering and chemical production.However,to date there is still lack of effective method to in-situ study chemical reaction dynamics for small molecule systems in solution[1].     

Ultrafast vibrational dynamics of interfacial water

We report investigations of the vibrational dynamics of water molecules at the water–air and at the water–lipid interface. Following vibrational excitation with an intense femtosecond infrared pulse resonant with the O–H stretch vibration of water, we follow the subsequent relaxation processes using the surface-specific spectroscopic technique of sum frequency generation. This allows us to selectively follow the vibrational relaxation of the approximately one monolayer of water molecules at the interface. Although the surface vibrational spectra of water at the interface with air and lipids are very similar, we find dramatic variations in both the rates and mechanisms of vibrational relaxation. For water at the water–air interface, very rapid exchange of vibrational energy occurs with water molecules in the bulk, and this intermolecular energy transfer process dominates the response. For membrane-bound water at the lipid interface, intermolecular energy transfer is suppressed, and intramolecular relaxation dominates. The difference in relaxation mechanism can be understood from differences in the local environments experienced by the interfacial water molecules in the two different systems.     

The chemical dynamics of nanosensors capable of single-molecule detection

Recent advances in nanotechnology have produced the first sensor transducers capable of resolving the adsorption and desorption of single molecules. Examples include near infrared fluorescent single-walled carbon nanotubes that report single-molecule binding via stochastic quenching. A central question for the theory of such sensors is how to analyze stochastic adsorption events and extract the local concentration or flux of the analyte near the sensor. In this work, we compare algorithms of varying complexity for accomplishing this by first constructing a kinetic Monte Carlo model of molecular binding and unbinding to the sensor substrate and simulating the dynamics over wide ranges of forward and reverse rate constants. Methods involving single-site probability calculations, first and second moment analysis, and birth-and-death population modeling are compared for their accuracy in reconstructing model parameters in the presence and absence of noise over a large dynamic range. Overall, birth-and-death population modeling was the most robust in recovering the forward rate constants, with the first and second order moment analysis very efficient when the forward rate is large (>10(-3) s(-1)). The precision decreases with increasing noise, which we show masks the existence of underlying states. Precision is also diminished with very large forward rate constants, since the sensor surface quickly and persistently saturates.     

Revealing time bunching effect in single-molecule enzyme conformational dynamics

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.     

Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics

Single-molecule F?rster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we can accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of nonfluorescent quenchers in single-molecule correlation studies.     

Architecturally diverse nanostructured foldamers reveal insightful photoinduced single-molecule dynamics

Single molecule fluorescence spectroscopy has been used to probe architecturally diverse and unique model oligomers containing exactly two or four perylene tetracarboxylic diimide (PTDI) units: linear foldamers lin2 and lin4, monocyclic complement cyc2, and concatenated foldable rings cat4. Linear, cyclic, and concatenated foldamers reveal that photoabsorption and excitation induces unfolding and refolding, generating colorful spectral switching from one spectral type to another. Foldamer architectures dictate the unfolding and refolding processes, and hence the spectral dynamics. As a result, linear tetramer exhibits active frame-to-frame spectral switching accompanying dramatic changes in colors, but a concatenated tetramer displays a multicolored composite spectrum with little or no spectral switching. Excited state dynamics causes spectral switching: an electronically decoupled PTDI monomer emits green fluorescence while electronically coupled PTDI pi-stacks emit red fluorescence, with longer pi-stacks emitting redder fluorescence. A key question we address is the excited-state delocalization length, or the exciton coherence length, in the pi-stacks, which has been proven difficult to measure directly. Using foldamers having controlled sequences, structures, and well-defined length and chromophore numbers, we have mapped out the exciton coherence length in pi-stacks. Single molecule fluorescence studies on chromophoric foldamers reveal that the maximum domain length is delocalized across just four pi-stacked PTDI dyes and no new pure color can be found for oligomers beyond the tetramer. Therefore, the range of fluorescent colors in pi-stacks is a function of the number of chromophores only up to the tetramer.     

Identifying ligand binding sites and poses using GPU-accelerated Hamiltonian replica exchange molecular dynamics

We present a method to identify small molecule ligand binding sites and poses within a given protein crystal structure using GPU-accelerated Hamiltonian replica exchange molecular dynamics simulations. The Hamiltonians used vary from the physical end state of protein interacting with the ligand to an unphysical end state where the ligand does not interact with the protein. As replicas explore the space of Hamiltonians interpolating between these states, the ligand can rapidly escape local minima and explore potential binding sites. Geometric restraints keep the ligands from leaving the vicinity of the protein and an alchemical pathway designed to increase phase space overlap between intermediates ensures good mixing. Because of the rigorous statistical mechanical nature of the Hamiltonian exchange framework, we can also extract binding free energy estimates for all putative binding sites. We present results of this methodology applied to the T4 lysozyme L99A model system for three known ligands and one non-binder as a control, using an implicit solvent. We find that our methodology identifies known crystallographic binding sites consistently and accurately for the small number of ligands considered here and gives free energies consistent with experiment. We are also able to analyze the contribution of individual binding sites to the overall binding affinity. Our methodology points to near term potential applications in early-stage structure-guided drug discovery.     

Segment dynamics in thin polystyrene films probed by single-molecule optics

Single fluorescent molecules (represented by spheres with a volume equal to the actual van der Waals volume of the molecule) has been embedded in a polystyrene matrix (left). Such molecules act as probes for the study of polymer nanoscale (segmental scale) dynamics in thin films deposited on a glass cover slide (right).     

Protein structure and dynamics from single-molecule fluorescence resonance energy transfer

The pros and cons of single-molecule vs ensemble-averaged fluorescence resonance energy transfer (FRET) experiments, performed on proteins, are explored with the help of Langevin dynamics simulations. An off-lattice model of the polypeptide chain is employed, which gives rise to a well-defined native state and two-state folding kinetics. A detailed analysis of the distribution of the donor-acceptor distance is presented at different points along the denaturation curve, along with its dependence on the averaging time window. We show that unique information on the correlation between structure and dynamics, which can only be obtained from single-molecule experiments, is contained in the correlation between the donor-acceptor distance and its displacement. The latter is shown to provide useful information on the free energy landscape of the protein, which is complementary to that obtained from the distribution of donor-acceptor distances.     

Probing the charge-transfer dynamics in DNA at the single-molecule level

Photoinduced charge-transfer fluorescence quenching of a fluorescent dye produces the nonemissive charge-separated state, and subsequent charge recombination makes the reaction reversible. While the information available from the photoinduced charge-transfer process provides the basis for monitoring the microenvironment around the fluorescent dyes and such monitoring is particularly important in live-cell imaging and DNA diagnosis, the information obtainable from the charge recombination process is usually overlooked. When looking at fluorescence emitted from each single fluorescent dye, photoinduced charge-transfer, charge-migration, and charge recombination cause a "blinking" of the fluorescence, in which the charge-recombination rate or the lifetime of the charge-separated state (τ) is supposed to be reflected in the duration of the off time during the single-molecule-level fluorescence measurement. Herein, based on our recently developed method for the direct observation of charge migration in DNA, we utilized DNA as a platform for spectroscopic investigations of charge-recombination dynamics for several fluorescent dyes: TAMRA, ATTO 655, and Alexa 532, which are used in single-molecule fluorescence measurements. Charge recombination dynamics were observed by transient absorption measurements, demonstrating that these fluorescent dyes can be used to monitor the charge-separation and charge-recombination events. Fluorescence correlation spectroscopy (FCS) of ATTO 655 modified DNA allowed the successful measurement of the charge-recombination dynamics in DNA at the single-molecule level. Utilizing the injected charge just like a pulse of sound, such as a "ping" in active sonar systems, information about the DNA sequence surrounding the fluorescent dye was read out by measuring the time it takes for the charge to return.     

Unfolding dynamics of cytochrome c revealed by single-molecule and ensemble-averaged spectroscopy

Denaturant-induced conformational change of yeast iso-1-cytochrome c (Cytc) has been comprehensively investigated in the single-molecule and bulk phases. By fluorescence-quenching experiments with dye-labelled heme-protein (Alexa 488-labelled Cytc, Cytc-A488), we clearly show that the fluorescence quenching observed from folded Cytc-A488 is due mainly to photoinduced electron transfer (PET) between electron-donating amino acids such as tryptophan and the dye attached to the protein. In addition, the unfolding process of Cytc-A488 observed in the single-molecule and bulk phases can be explained well in terms of a three-state model: Cytc unfolds through an intermediate with a native-like compactness. By quantitative analysis of fluorescence correlation spectroscopy (FCS) data, we were able to observe a relaxation time of ～1.5 μs corresponding to segmental motion and fast folding dynamics of 55 μs in the unfolded state of Cytc. The results presented here also suggest that a combination of single-molecule and ensemble-averaged spectroscopy is necessary to provide convincing and comprehensive assignments of protein kinetics.     

Extracting the time scales of conformational dynamics from single-molecule single-photon fluorescence statistics

The quenching rate of a fluorophore attached to a macromolecule can be rather sensitive to its conformational state. The decay of the corresponding fluorescence lifetime autocorrelation function can therefore provide unique information on the time scales of conformational dynamics. The conventional way of measuring the fluorescence lifetime autocorrelation function involves evaluating it from the distribution of delay times between photoexcitation and photon emission. However, the time resolution of this procedure is limited by the time window required for collecting enough photons in order to establish this distribution with sufficient signal-to-noise ratio. Yang and Xie have recently proposed an approach for improving the time resolution, which is based on the argument that the autocorrelation function of the delay time between photoexcitation and photon emission is proportional to the autocorrelation function of the square of the fluorescence lifetime [Yang, H.; Xie, X. S. J. Chem. Phys. 2002, 117, 10965]. In this paper, we show that the delay-time autocorrelation function is equal to the autocorrelation function of the square of the fluorescence lifetime divided by the autocorrelation function of the fluorescence lifetime. We examine the conditions under which the delay-time autocorrelation function is approximately proportional to the autocorrelation function of the square of the fluorescence lifetime. We also investigate the correlation between the decay of the delay-time autocorrelation function and the time scales of conformational dynamics. The results are demonstrated via applications to a two-state model and an off-lattice model of a polypeptide.     

Conformational dynamics of the isoalloxazine in substrate-free p-hydroxybenzoate hydroxylase: single-molecule studies

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric enzyme in which each subunit noncovalently binds one molecule of FAD in the active site. PHBH is a model system for how flavoenzymes regulate reactions with oxygen. We report single-molecule fluorescence studies of PHBH in the absence of substrate that provide data consistent with the hypothesis that a critical step in substrate binding is the movement of the isoalloxazine between an "in" conformation and a more exposed or "open" conformation. The isoalloxazine is observed to move between these conformations in the absence of substrate. Studies with the Y222A mutant form of PHBH suggest that the exposed conformation is fluorescent while the in-conformation is quenched. Finally, we note that many of the single-molecule-fluorescence trajectories reveal a conformational heterogeneity, with populations of the enzyme characterized by either fast or slow switching between the in- and open-conformations. Our data also allow us to hypothesize a model in which one flavin in the dimer inhibits the motion of the other.     

Probing irradiation induced DNA damage mechanisms using excited state Car-Parrinello molecular dynamics

Photoinduced proton transfer in the Watson-Crick guanine (G)-cytosine (C) base pair has been studied using Car-Parrinello molecular dynamics (CP-MD). A flexible mechanical constraint acting on all three hydrogen bonds in an unbiased fashion has been devised to explore the free energy profile along the proton transfer coordinate. The lowest barrier has been found for proton transfer from G to C along the central hydrogen bond. The resulting charge transfer excited state lies energetically close to the electronic ground state suggesting the possibility of efficient radiationless decay. It is found that dynamic, finite temperature fluctuations significantly reduce the energy gap between the ground and excited states for this charge transfer product, promoting the internal conversion process. A detailed analysis of the internal degrees of freedom reveals that the energy gap is considerably reduced by out-of-plane molecular vibrations, in particular. Consequently, it appears that considering only the minimum energy path provides an upper-bound estimate of the associated energy gap compared to the full-dimension dynamical reaction coordinate. Furthermore, the first CP-MD simulations of the G-C base pair in liquid water are presented, and the effects of solvation on its electronic structure are analyzed.