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N-methyl-d-aspartate (NMDA) receptor ion channel is activated by the binding of two pairs of glycine and glutamate along with the application of action potential. Binding and unbinding of ligands changes its conformation that plays a critical role in the open-close activities of NMDA receptor. Conformation states and their dynamics due to ligand binding are extremely difficult to characterize either by conventional ensemble experiments or single-channel electrophysiology method. Here we report the development of a new correlated technical approach, single-molecule patch-clamp FRET anisotropy imaging and demonstrate by probing the dynamics of NMDA receptor ion channel and kinetics of glycine binding with its ligand binding domain. Experimentally determined kinetics of ligand binding with receptor is further verified by computational modeling. Single-channel patch-clamp and four-channel fluorescence measurement are recorded simultaneously to get correlation among electrical on and off states, optically determined conformational open and closed states by FRET, and binding-unbinding states of the glycine ligand by anisotropy measurement at the ligand binding domain of GluN1 subunit. This method has the ability to detect the intermediate states in addition to electrical on and off states. Based on our experimental results, we have proposed that NMDA receptor gating goes through at least one electrically intermediate off state, a desensitized state, when ligands remain bound at the ligand binding domain with the conformation similar to the fully open state.

Fundamental understanding of the energetic coupling properties of a molecule-semiconductor interface is of great importance. The changes in molecular conformations and vibrational modes can have significant impact on the interfacial charge transfer reactions. Here, we have probed the change in the interface properties of alizarin-TiO2 system as a result of the externally applied electric field using single-hot spot microscopic surface-enhanced Raman spectroscopy (SMSERS) and provided a theoretical understanding of our experimental results by density functional theory (DFT) calculations. The perturbation, caused by the external potential, has been observed as a shift and splitting of the 648 cm(-1) peak, typical indicator of the strong coupling between alizarin and TiO2, at SMSERS. On the basis of our experimental results and DFT calculations, we suggest that electric field has significant effects on vibrational coupling at the molecule-TiO2 interface. The presence of perturbed alizarin-TiO2 coupling under interfacial electric potential may lead to changes in the interfacial electron transfer dynamics. Additionally, heterogeneously distributed dye molecules at the interface on nanometer length scale and different chromophore-semiconductor binding interactions under charge accumulation associated interfacial electric field changes create intrinsically inhomogeneous interfacial ET dynamics associated with both static and dynamic disorders.

Conformational dynamics plays a critical role in the activation, deactivation, and open-close activities of ion channels in living cells. Such conformational dynamics is often inhomogeneous and extremely difficult to be directly characterized by ensemble-averaged spectroscopic imaging or only by single channel patch-clamp electric recording methods. We have developed a new and combined technical approach, single-molecule patch-clamp FRET microscopy, to probe ion channel conformational dynamics in living cell by simultaneous and correlated measurements of real-time single-molecule FRET spectroscopic imaging with single-channel electric current recording. Our approach is particularly capable of resolving ion channel conformational change rate process when the channel is at its electrically off states and before the ion channel is activated, the so-called "silent time" when the electric current signals are at zero or background. We have probed NMDA (N-methyl-D-aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open-close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. On the basis of our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open-close dynamics.

The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. Using single-molecule fluorescence resonance energy transfer (FRET) with a combined statistical data analysis approach, we have identified the intermittently appearing coherence of the enzymatic conformational state from the recorded single-molecule intensity-time trajectories of enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in catalytic reaction. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multistep conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. The coherence frequency, identified by statistical results of the correlation function analysis from single-molecule FRET trajectories, increases with the increasing substrate concentrations. The intermittent coherence in conformational state changes at the enzymatic reaction active site is likely to be common and exist in other conformation regulated enzymatic reactions. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

We have probed single-molecule redox reaction dynamics of hemin (chloride) adsorbed on Ag nanoparticle surfaces by single-molecule surface-enhanced Raman spectroscopy (SMSERS) combined with spectroelectrochemistry. Redox reaction at the molecule/Ag interface is identified and probed by the prominent fluctuations of the Raman frequency of a specific vibrational mode, ν(4), which is a typical marker of the redox state of the iron center in a hemin molecule. On the basis of the autocorrelation and cross-correlation analysis of the single-molecule Raman spectral trajectories and the control measurements of single-molecule spectroelectochemistry and electrochemical STM, we suggest that the single-molecule redox reaction dynamics at the hemin-Ag interface is primarily driven by thermal fluctuations. The spontaneous fluctuation dynamics of the single-molecule redox reaction is measured under no external electric potential across the molecule-metal interfaces, which provides a novel and unique approach to characterize the interfacial electron transfer at the molecule-metal interfaces. Our demonstrated approaches are powerful for obtaining molecular coupling and dynamics involved in interfacial electron transfer processes. The new information obtained is critical for a further understanding, design, and manipulation of the charge transfer processes at the molecule-metal interface or metal-molecule-metal junctions, which are fundamental elements in single-molecule electronics, catalysis, and solar energy conversion., (© 2011 American Chemical Society)

The inhomogeneous interfacial electron transfer (IET) dynamics of 9-phenyl-2,3,7-trihydroxy-6-fluorone (PF)-sensitized TiO(2) nanoparticles (NPs) has been probed by a single-molecule photon-stamping technique as well as ensemble-averaged femtosecond transient absorption spectroscopy. The forward electron transfer (FET) time shows a broad distribution at the single-molecule level, indicating the inhomogeneous interactions and ET reactivity of the PF/TiO(2) NP system. The broad distribution of the FET time is measured to be 0.4 +/- 0.1 ps in the transient absorption and picoseconds to nanoseconds in the photon-stamping measurements. The charge recombination time, having a broad distribution at the single-molecule level, clearly shows a biexponential dynamic behavior in the transient absorption: a fast component of 3.0 +/- 0.1 ps and a slow component of 11.5 +/- 0.5 ns. We suggest that both strong and weak interactions between PF and TiO(2) coexist, and we have proposed two mechanisms to interpret the observed IET dynamics. A single-molecule photon-stamping technique and ensemble-averaged transient absorption spectroscopy provide efficient "zoom-in" and "zoom-out" approaches for probing the IET dynamics. The physical nature of the observed multiexponential or stretched-exponential ET dynamics in the ensemble-averaged experiments, often associated with dynamic and static inhomogeneous ET dynamics, can be identified and analyzed by single-molecule spectroscopy measurements.

Interfacial electron-cation recombination in zinc-tetra (4-carboxyphenyl) porphyrin (ZnTCPP)/TiO(2) nanoparticle system has been probed at the single-molecule level by recording and analyzing photon-to-photon pair times of the ZnTCPP fluorescence. We have developed a novel approach to reveal the hidden single-molecule interfacial electron-cation recombination dynamics by analyzing the autocorrelation function and a proposed convoluted single-molecule interfacial electron-cation recombination model. Our results suggest that the fluctuations of the interfacial electron transfer (ET) reactivity modulate the ET cycles as well as the interfacial electron-cation recombination dynamics. On the basis of this model, the single-molecule electron-cation recombination time of ZnTCPP/TiO(2) system is deduced to be at time scale of 10(-5) s. The autocorrelation of photon-to-photon pair times as well as the convoluted ET model has been further demonstrated by simulation and interpreted in terms of the interfacial ET reactivity fluctuation and blinking. Our approach not only can effectively probe the single-molecule interfacial electron-cation dynamics but also can be applied to other single-molecule ground-state regeneration dynamics occurring at interfaces and within condensed phases.

Single-molecule interfacial electron transfer (ET) dynamics has been studied by using single-molecule fluorescence spectroscopy and microscopic imaging. For a single-molecule zinc-tetra (4-carboxyphenyl) porphyrin (ZnTCPP)/TiO(2) nanoparticle system, the single-molecule fluorescence trajectories show strong fluctuation and blinking between bright and dark states. The intermittency and fluctuation of the single-molecule fluorescence are attributed to the variation of the reactivity of interfacial electron transfer. The nonexponential autocorrelation function and the power-law distribution of the probability density of dark times imply the dynamic and static inhomogeneities of the interfacial ET dynamics. On the basis of the power-law analysis, the variation of single-molecule interfacial ET reactivity is analyzed as a fluctuation according to the Levy statistics.

We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM/C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (FlAsH) that enables our unambiguous probing of the CaM N-terminal target-binding domain motions on a millisecond time scale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between FlAsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal binding-unbinding motions of the N-terminal domain of the CaM in CaM/C28W complexes, which is strong evidence of a two-state binding interaction of CaM-mediated cell signaling.

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.