Biomedical subjects

H Peter Lu

Publications and source records attributed to H Peter Lu.

8 recordsLinked to original sources

PubMed2005

Probing single-molecule protein conformational dynamics.

Protein conformational fluctuations and dynamics, often complex and associated with inhomogeneities, play a crucial role in biomolecular functions. It is extremely difficult to characterize such inhomogeneous dynamics in an ensemble-averaged measurement, especially when the proteins are involved in multiple-step, multiple-conformation complex chemical interactions and transformations, such as in enzymatic reactions, protein-protein interactions, and ion-channel membrane protein processes. Alternatively, single-molecule spectroscopy is a powerful approach to probing and analyzing protein conformational dynamics in real time.

Bacteriophage T4↗

PubMed2005

Single-molecule study of protein-protein and protein-DNA interaction dynamics.

Protein-protein and protein-DNA interactions play critical roles in biological systems, and these interactions often involve complex mechanisms and inhomogeneous dynamics. Single-molecule spectroscopy is a powerful and complimentary approach to decipher such spatially and temporally inhomogeneous protein interaction systems, providing new information that are not obtainable from static structure analyses, thermodynamics characterization, and ensemble-averaged measurements. To illustrate the single-molecule spectroscopy and imaging technology and their applications on studying protein-ligand interactions, this chapter focuses on discussing two recent single-molecule spectroscopy studies on protein-protein interaction in cell signaling process and on protein-DNA interactions in DNA damage recognition process.

Binding Sites↗

PubMed2004

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.

Journal Article↗

PubMed2004

Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells.

We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM). For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly-L-lysine coated surfaces or agarose gel coated surfaces. We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and signal-to-noise ratio of the AFM images. Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM-FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.

Bacteria↗

PubMed2004

Placing single-molecule T4 lysozyme enzymes on a bacterial cell surface: toward probing single-molecule enzymatic reaction in living cells.

The T4 lysozyme enzymatic hydrolyzation reaction of bacterial cell walls is an important biological process, and single-molecule enzymatic reaction dynamics have been studied under physiological condition using purified Escherichia coli cell walls as substrates. Here, we report progress toward characterizing the T4 lysozyme enzymatic reaction on a living bacterial cell wall using a combined single-molecule placement and spectroscopy. Placing a dye-labeled single T4 lysozyme molecule on a targeted bacterial cell wall by using a hydrodynamic microinjection approach, we monitored single-molecule rotational motions during binding, attachment to, and dissociation from the cell wall by tracing single-molecule fluorescence intensity time trajectories and polarization. The single-molecule attachment duration of the T4 lysozyme to the cell wall during enzymatic reactions was typically shorter than the photobleaching time under physiological conditions. Applying single-molecule fluorescence polarization measurements to characterize the binding and motions of the T4 lysozyme molecules, we observed that the motions of wild-type and mutant T4 lysozyme proteins are essentially the same whether under an enzymatic reaction or not. The changing of the fluorescence polarization suggests that the motions of the T4 lysozyme are associated with orientational rotations. This observation also suggests that the T4 lysozyme binding-unbinding motions on cell walls involve a complex mechanism beyond a single-step first-order rate process.

Bacteriophage T4↗

PubMed2004

Single-molecule spectroscopy studies of conformational change dynamics in enzymatic reactions.

Protein conformational dynamics, often associated with static and dynamic inhomogeneities, plays a crucial role in biomolecular functions. It is extremely difficult to characterize such inhomogeneous dynamics in an ensemble-averaged measurement, especially when the protein involves in a multiple-step complex chemical reaction, such as an enzymatic reaction. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics in real time under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. In this minireview, we discuss our recent studies on single-molecule enzymatic reaction dynamics and protein conformational dynamics of the T4 lysozyme hydrolyzation reaction of a polysaccharide by a combined approach of single-molecule spectroscopy, molecular dynamics simulation, and theoretical modeling. Detailed characterization of the complex enzymatic reaction dynamics identified the nature of the inhomogeneity and revealed multiple intermediate conformational states associated with the enzyme-substrate complex formation in the multiple-step enzymatic reaction.

Animals↗

PubMed2003

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.

Anti-Bacterial Agents↗

PubMedDate not supplied

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.

Journal Article↗