H. Peter Lu

Probing Conformational Changes of Gramicidin Ion Channels by Single-Molecule Patch-Clamp Fluorescence Microscopy

Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.

Probing Single-Molecule Interfacial Electron Transfer Dynamics of Porphyrin on TiO2 Nanoparticles

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.

Probing single-molecule enzyme active-site conformational state intermittent coherence.

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.

Probing nanoscale surface enhanced Raman-scattering fluctuation dynamics using correlated AFM and confocal ultramicroscopy

We have studied the laser-excitation-intensity-dependent and Ag-nanocluster interstitial-site-dependent SERS intensity fluctuations under low molecule surface coverage of rhodamine 6G and cytochrome c. A new two-channel photon time-stamping system coupled with atomic force microscopic (AFM), Raman spectroscopic, and imaging microscopy was developed and applied to record Raman intensity fluctuation trajectories at sub-microsecond resolution correlated with in situ characterization of the nanoparticle clusters. Our experimental results suggest that the nanoconfinement of the local electromagnetic-field enhancement and the interaction of the local field with the molecules, presumably under rotational motions, result in nano-Raman fluctuations. The SERS spectral fluctuation was pertinent to the nanoscale local enhancement and local interaction of the molecules with the surface when the surface coverage of the nanoparticles was less than a monolayer, and the nanoscale interstitial space controlled the finite number of molecules to contribute the microscopic Raman signal collected from a diffraction-limited focus spot. The fluctuation amplitude significantly decreased with the number of molecules confined at the nanolocal field. The nano-SERS fluctuation dynamics were both photo-induced and spontaneous for rhodamine 6G, but only the photo-induced component was observable for cytochrome c. The fluctuation dynamics were also found to be highly inhomogeneous at interstitial sites with heterogeneous geometries. To interpret the observed nano-SERS fluctuation dynamics, we used computer simulation of optical multiple scattering, based on multi-sphere scattering Mie theory, and rotational diffusion of molecules at an interstitial site, based on a random walk in orientation space.

Single-molecule dynamics reveals cooperative binding-folding in protein recognition

The study of associations between two biomolecules is the key to understanding molecular function and recognition. Molecular function is often thought to be determined by underlying structures. Here, combining a single-molecule study of protein binding with an energy-landscape–inspired microscopic model, we found strong evidence that biomolecular recognition is determined by flexibilities in addition to structures. Our model is based on coarse-grained molecular dynamics on the residue level with the energy function biased toward the native binding structure (the Go model). With our model, the underlying free-energy landscape of the binding can be explored. There are two distinct conformational states at the free-energy minimum, one with partial folding of CBD itself and significant interface binding of CBD to Cdc42, and the other with native folding of CBD itself and native interface binding of CBD to Cdc42. This shows that the binding process proceeds with a significant interface binding of CBD with Cdc42 first, without a complete folding of CBD itself, and that binding and folding are then coupled to reach the native binding state. The single-molecule experimental finding of dynamic fluctuations among the loosely and closely bound conformational states can be identified with the theoretical, calculated free-energy minimum and explained quantitatively in the model as a result of binding associated with large conformational changes. The theoretical predictions identified certain key residues for binding that were consistent with mutational experiments. The combined study identified fundamental mechanisms and provided insights about designing and further exploring biomolecular recognition with large conformational changes.

Tip-Enhanced Near-Field Raman Spectroscopy Probing Single Dye-Sensitized TiO2 Nanoparticles

The correlated metallic tip-enhanced Raman spectroscopy and atomic force microscopy (AFM) technique was used to characterize dye-sensitized titanium oxide (TiO2) nanoparticles. We have obtained the near-field Raman spectra that are associated with the photo-induced charge transfer reaction in Ru(4,4’-dicarboxy-2,2’-bipyridine)2(NCS)2-sensitized TiO2 single nanoparticles. This method demonstrates that tip-enhanced near-field Raman spectroscopy is an effective approach for understanding inhomogeneous interfacial electron transfers with nanoscale spatial resolution.

Probing Single-Molecule Interfacial Geminate Electron―Cation Recombination Dynamics

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.

Correlated topographic and spectroscopic imaging beyond diffraction limit by atomic force microscopy metallic tip-enhanced near-field fluorescence lifetime microscopy

A near-field optical imaging approach is demonstrated for simultaneous topographic and spectroscopic imaging with spatial resolution beyond the optical diffraction limit. The method combines metallic-tip-based tapping-mode atomic force microscopy (AFM) with fluorescence lifetime imaging microscopy (FLIM). The AFM metallic tip was formed by sputter coating a Si tapping mode tip with Au, in a way that forms a globular tip apex. Such tip apex generates high local electric field enhancement under laser illumination, which provides a strong electric-field interaction between the AFM tip and the fluorescent molecules under the tip. The tip perturbation of fluorescence gives the fluorescence lifetime changes that provide the AFM–FLIM imaging contrast. A finite element method simulation was used to further evaluate the electric near-field enhancement and electric field distribution originating from the metallic Au-coated AFM tapping-mode tip. We have demonstrated that spatially mapping the change in fluorescence ...

Probing Ground-state Single-electron Self-exchange Across A Molecule-metal Interface

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.

Probing ion channel conformational dynamics using simultaneous single-molecule ultrafast spectroscopy and patch-clamp electric recording

An approach to probing single-molecule ion channel kinetics and conformational dynamics, patch-clamp confocal fluorescence microscopy (PCCFM), uses simultaneous ultrafast fluorescence spectroscopy and single-channel electric current recording. PCCFM is applied to determine single-channel conformational dynamics by probing single-pair fluorescence resonant energy transfer, fluorescence self-quenching, and anisotropy of the dye-labeled gramicidin ion channel incorporated in an artificial lipid bilayer. Hidden conformational changes were observed, which strongly suggests that multiple intermediate conformation states are involved in gramicidin ion channel dynamics.