Cover image for Vol. 6 Issue 5

Special Issue: Single-Molecule Studies and Biomolecular Dynamics

May 13, 2005

Volume 6, Issue 5

Pages 753–998

    1. Cover Picture: Single-Molecule Quantum-Dot Fluorescence Resonance Energy Transfer (ChemPhysChem 5/2005) (page 753)

      Sungchul Hohng and Taekjip Ha

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200590006

      The cover picture shows fluorescence resonance energy transfer (FRET) between single-molecule quantum dots (green) as donors and organic acceptors (red). The conformational dynamics of a DNA four-way junction tagged with this FRET pair are observed. Recent discovery of quantum-dot blinking suppression opened up new opportunities to observe single-molecule conformational changes using long-lasting, ultrabright quantum dots. In their article on page 956 T. Ha and S. Hohng show that single-molecule structural dynamics can be reliably probed using these quantum dots.

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      Editorial: The Coming of Age (pages 755–758)

      Gilad Haran and Peter Vöhringer

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200500140

    3. Statistical Evaluation of Single Nano-Object Fluorescence (pages 770–789)

      Markus Lippitz, Florian Kulzer and Michel Orrit

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400560

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      Random blinking of single molecules: The photon stream emitted by a single nano-object must be statistically evaluated to provide microscopic information. The authors compare methods used to gain this information, illustrated by simulated data, and review experiments performed using such methods on different types of nano-objects (see scatter plots of a single-enzyme model).

    4. Molecular Photobleaching Kinetics of Rhodamine 6G by One- and Two-Photon Induced Confocal Fluorescence Microscopy (pages 791–804)

      Christian Eggeling, Andreas Volkmer and Claus A. M. Seidel

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400509

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      Photobleaching single Rhodamine 6G molecules: The authors determine photokinetic parameters and disclose the underlying photobleaching mechanisms of Rhodamine 6G (see picture; NF=number of photon counts per observation time) under high-excitation irradiance conditions (I0/2) by one- and two-photon induced fluorescence microscopy.

    5. Intramolecular Electron Transfer in Nitrite Reductases (pages 805–812)

      Scot Wherland, Ole Farver and Israel Pecht

      Version of Record online: 1 APR 2005 | DOI: 10.1002/cphc.200400353

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      Multimeric enzymes: Nitrite reductases (NiRs) contain both an electron-accepting site and a site at which nitrite is reduced to nitric oxide in each monomer. The sites can be either copper or heme iron, and there are significant structural differences even within these families. The picture shows the electron transfer (ET) pathway between the two Cu sites of the NiR from A. faecalis.

    6. Single-Molecule Manipulation Measurements of DNA Transport Proteins (pages 813–818)

      Omar A. Saleh, Jean-François Allemand, Vincent Croquette and David Bensimon

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400635

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      Driving DNA along: Single-molecule measurements of the manipulation of DNA motor proteins are reviewed in terms of the different available experimental approaches and the results that they provide. Using three motor proteins as examples, differences in transport mechanisms and structures are discussed. A schematic representation of a measurement on FtsK is presented in the figure.

    7. Energy Transfer in Single-Molecule Photonic Wires (pages 819–827)

      María F. García-Parajó, Jordi Hernando, Gabriel Sanchez Mosteiro, Jacob P. Hoogenboom, Erik M. H. P. van Dijk and Niek. F. van Hulst

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400630

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      Molecular photonics is a new emerging field of research around the premise that it is possible to develop optical devices using single molecules as building blocks (see picture). In this Minireview the authors discuss three different approaches towards the design and single-molecule investigation of one-molecular photonic wires.

    8. Initial Photoinduced Dynamics of the Photoactive Yellow Protein (pages 828–837)

      Delmar S. Larsen and Rienk van Grondelle

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400351

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      Probing the yellow protein photocycle: Light absorption by the intrinsic chromophore within the photoactive yellow protein (PYP; see picture) leads to the initiation of a photocycle, which evolves on multiple timescales and includes several distinct intermediates. The dynamic processes responsible for the initiation of the PYP photocycle are explored with different time-resolved techniques, such as ultrafast electronic and vibrational spectroscopies.

    9. Green and Red Fluorescent Proteins: Photo- and Thermally Induced Dynamics Probed by Site-Selective Spectroscopy and Hole Burning (pages 838–849)

      S. Bonsma, R. Purchase, S. Jezowski, J. Gallus, F. Könz and S. Völker

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200500005

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      Green and red fluorescent proteins are favorite markers for tracking motions and protein–protein interactions in live cells. This Minireview surveys the elusive photophysics of green wt-GFP and red DsRed (see picture) by site-selective spectroscopy and hole burning at liquid-helium temperature and examines the changes that occur upon raising the temperature. The physical implications of the observed photoconversions, energy-transfer and dephasing processes are discussed.

    10. Coherent Control for Spectroscopy and Manipulation of Biological Dynamics (pages 850–857)

      Wendel Wohlleben, Tiago Buckup, Jennifer L. Herek and Marcus Motzkus

      Version of Record online: 1 APR 2005 | DOI: 10.1002/cphc.200400414

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      Control of quantum phenomena in complex molecules has been difficult. With feedback-optimized coherent control, the authors successfully steered the energy flow in the antenna complex LH2 of Rps. acidophila with an optimal 160 cm−1 modulation (see picture). In their Minireview, the authors strive to give an overall picture of the events in LH2 when excited with shaped pulses.

    11. The Study of Protein Folding and Dynamics by Determination of Intramolecular Distance Distributions and Their Fluctuations Using Ensemble and Single-Molecule FRET Measurements (pages 858–870)

      Elisha Haas

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400617

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      Tricky proteins: After almost 50 years of protein-folding research we are still unable to read genes. Determination of intramolecular distance distributions by ensemble and single-molecule FRET experiments (see picture) enabled the author to explore the partially folded states of refolding protein molecules. One observation is that some proteins start the folding transition by formation of a few long loops and thus reduce the chain entropy at the start.

    12. The First Picoseconds in Bacterial Photosynthesis—Ultrafast Electron Transfer for the Efficient Conversion of Light Energy (pages 871–880)

      Wolfgang Zinth and Josef Wachtveitl

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400458

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      The first photochemical processes in photosynthesis occur in the photosynthetic reaction centre (RC). After excitation of the primary donor (P) the light energy is converted into chemical energy by ultrafast charge separation followed by electron transfer over a chain of neighbouring chromophores (see graphic; BA, HA and QA are the accessory bacteriochlorophyll, bacteriopheophytin and quinone groups).

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      Proteins in Action Monitored by Time-Resolved FTIR Spectroscopy (pages 881–888)

      Carsten Kötting and Klaus Gerwert

      Version of Record online: 6 APR 2005 | DOI: 10.1002/cphc.200400504

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      Protein reactions: Time-resolved Fourier-transform infrared (trFTIR) difference spectroscopy is a method that is capable of providing useful molecular detail of the mechanisms underlying protein reactions (see picture). The main principles of the technique are described, including a summary of triggering techniques, scan modes and analysis, together with an overview of applications investigated by the authors.

    14. Carriers versus Adapters in Stochastic Sensing (pages 889–892)

      Orit Braha, Jadon Webb, Li-Qun Gu, Kimoon Kim and Hagan Bayley

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400595

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      The perfect host: The detection of analyte-dependent changes in the conduction of individual transmembrane protein pores is demonstrated for small organic molecules, which are detected as complexes with a carrier molecule, cucurbit[6]uril (CB6). The carrier system also allows the determination of dissociation constants, and association and dissociation rate constants, for CB6⋅guest pairs (see picture; αHL=α-hemolysin, THF=tetrahydrofuran).

    15. Two-Photon Absorption of Bis[4-(N,N-diphenylamino)phenylethynyl]arenes (pages 893–896)

      Bernd Strehmel, Stephan Amthor, Jürgen Schelter and Christoph Lambert

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400538

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      Attractive chromophores for photoscience: Two-photon (TP) absorption (TPA) of ethynyl chromophores 25 (see picture) was studied using femtosecond excitation between 710–950 nm. Compounds 25 possess higher excitation energies for the TP-excited than for the one-photon (OP) excited states. Chromophore 2 exhibits an additional slight absorption band at the S0[RIGHTWARDS ARROW]S1 transition. This is explained by a vibronic coupling mechanism.

    16. Localization of Single Avidin–Biotin Interactions Using Simultaneous Topography and Molecular Recognition Imaging (pages 897–900)

      Andreas Ebner, Ferry Kienberger, Gerald Kada, Cordula M. Stroh, Manfred Geretschläger, A. S. M. Kamruzzahan, Linda Wildling, W. Travis Johnson, Brian Ashcroft, Jeremy Nelson, Stuart M. Lindsay, Hermann J. Gruber and Peter Hinterdorfer

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400545

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      Two-for-one: The authors discuss a powerful new tool that, in a single dynamic force microscopy scan, maps the topography of a surface and simultaneously recognizes specific target sites (see figure). Topography and recognition images correlated nicely at a lateral resolution of a few nanometers. This clever technique has great potential for a broad range of applications involving the detection of particular binding sites in native biological systems.

    17. Nanopatterning of Biomolecules with Microscale Beads (pages 900–903)

      Patrick Pammer, Robert Schlapak, Max Sonnleitner, Andreas Ebner, Rong Zhu, Peter Hinterdorfer, Otmar Höglinger, Hansgeorg Schindler and Stefan Howorka

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400526

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      Nanopatterning of biomolecules: Arrays of microscale beads can be used as stamps to print biomolecules onto a flat substrate. The simple surface-structuring method produces regular patterns of nanoscale DNA and protein spots with a diameter of 300 nm separated by an interspot distance of several micrometers (see figure).

    18. Effect of Bin Time on the Photon Counting Histogram for One-Photon Excitation (pages 905–912)

      Thomas D. Perroud, Bo Huang and Richard N. Zare

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400547

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      The photon counting histogram (PCH) model with the correction for one-photon excitation is valid at multiple bin times. More importantly, the semi-empirical parameter, F, introduced in the PCH model for one-photon excitation to correct for the non-Gaussian shape of the observation volume, shows small variations with different bin times (see picture; ε=molecular brightness; N=number of particles). These variations are consistent with the physical interpretation of F, and they do not affect the resolving power of the PCH model for one-photon excitation.

    19. Surface-Enhanced Raman Scattering of p-Aminothiophenol on a Au(core)/Cu(shell) Nanoparticle Assembly (pages 913–918)

      Linyou Cao, Peng Diao, Lianming Tong, Tao Zhu and Zhongfan Liu

      Version of Record online: 31 MAR 2005 | DOI: 10.1002/cphc.200400254

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      Chemical enhancement in surface-enhanced Raman scattering of p-aminothiophenol (PATP) on Au(core)/Cu(shell) particles (see picture) may be identified quantitatively by comparison with the Raman spectrum of PATP on gold particles. The enhancement is associated with the density of states and the Fermi level of the metal substrates, as shown by theoretical analysis. Electromagnetic enhancement on these core/shell particles is also shown to be different from those of both gold and copper substrates.

    20. Vibronic Excitation of Single Molecules: A New Technique for Studing Low-Temperature Dynamics (pages 919–925)

      Alper Kiraz, Moritz Ehrl, Christian Hellriegel, Christoph Bräuchle and Andreas Zumbusch

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400579

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      Jumps in the cold: Optical spectroscopy of single molecules at cryogenic temperatures gives insight into dynamics of solids, commonly modelled by two-level system transitions. Excitation of vibronic chromophore bands and detection of narrow zero-phonon lines are new techniques for such investigations. They allow the observation and analysis of hitherto unknown large spectral jumps of emission bands in dye-doped crystals and polymers (see picture).

    21. Single Chromophore Spectroscopy of MEH-PPV: Homing-In on the Elementary Emissive Species in Conjugated Polymers (pages 926–934)

      Florian Schindler and John M. Lupton

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400575

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      Single-molecule studies on polymers: The authors present results on high-resolution fluorescence spectroscopy of highly disordered poly(2-methoxy-5-(2′-ethylhexoxy)-1,4-phenylenevinylene). A multitude of low-frequency, vibrationally active modes are identified along with pronounced zero-phonon lines. The spectral identification of single chromophores (see figure) allows a direct comparison of the homogeneous and inhomogeneous contributions to spectral broadening in complex systems.

    22. Photophysical Properties of a Tetraphenoxy-Substituted Perylene Bisimide Derivative Characterized by Single-Molecule Spectroscopy (pages 935–941)

      Erwin Lang, Frank Würthner and Jürgen Köhler

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400555

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      Perylene bisimide molecules are of great interest for artificial light harvesting and as photoluminescent organic materials. The authors present the characterization of the photophysical properties of a perylene bisimide derivative by single-molecule spectroscopic techniques at cryogenic temperatures (see figure).

    23. Electron Transfer at the Single-Molecule Level in a Triphenylamine–Perylene Imide Molecule (pages 942–948)

      Toby D. M. Bell, Alina Stefan, Sadahiro Masuo, Tom Vosch, Marc Lor, Mircea Cotlet, Johan Hofkens, Stefan Bernhardt, Klaus Müllen, Mark van der Auweraer, Jan W. Verhoeven and Frans C. De Schryver

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400567

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      Electron transfer in single molecules is difficult to study as this process usually quenches fluorescence emission. The molecular system presented here (see picture) undergoes delayed fluorescence in toluene and polystyrene, which allows the electron-transfer (ET) equilibrium to be studied in detail. In polystyrene, single molecules show a wide range of behaviours, with frequent long-lived “dim-states” and “off-states” being observed and attributable to successive cycles of photoinduced ET.

    24. High-Resolution Colocalization of Single Molecules within the Resolution Gap of Far-Field Microscopy (pages 949–955)

      Thomas Heinlein, Andreas Biebricher, Pia Schlüter, Christian Michael Roth, Dirk-Peter Herten, Jürgen Wolfrum, Mike Heilemann, Christian Müller, Philip Tinnefeld and Markus Sauer

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400622

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      GPS for single molecules: The determination of the absolute three-dimensional localization of individual molecules and their intermolecular distances in small biomolecular assemblies requires a kind of “cellular positioning system” (CPS; see picture). The authors demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) in combination with polarization-modulated excitation represents a versatile technique to determine distances between single molecules far below the optical resolution limit and to measure their relative orientations.

    25. Single-Molecule Quantum-Dot Fluorescence Resonance Energy Transfer (pages 956–960)

      Sungchul Hohng and Taekjip Ha

      Version of Record online: 1 APR 2005 | DOI: 10.1002/cphc.200400557

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      Single-molecule dynamics: Semiconductor quantum dots can be used for studies of single-molecule dynamics. The authors used a quantum dot (QD) as donor and an organic acceptor for fluorescence resonance energy transfer (FRET) measurements and observed conformational dynamics from a DNA four-way junction tagged with this FRET pair (picture, top). The time trace of this composite shows anticorrelated fluctuations of signals in the donor (green) and acceptor (red) channels (bottom).

    26. Electrochemical and Spectroscopic Investigations of Immobilized De Novo Designed Heme Proteins on Metal Electrodes (pages 961–970)

      Tim Albrecht, Wenwu Li, Jens Ulstrup, Wolfgang Haehnel and Peter Hildebrandt

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400597

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      De novo designed heme proteins offer new possibilities for the design of tailor-made immobilized enzymes. The authors have synthesized heme proteins that exhibit different binding domains for attachment to metal electrodes (see figure). The structures and redox equilibria, as well as the dynamics of the immobilized proteins, were studied by surface-enhanced resonance Raman spectroscopy and electrochemical methods.

    27. Vibronic Transitions in Single Metalloporphyrins (pages 971–975)

      H. J. Lee, J. H. Lee and W. Ho

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400616

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      Electron–vibration coupling: A low-temperature scanning tunneling microscope was used to probe the conformation-dependent vibronic states of individual zinc etioporphyrin molecules on Al2O3/Ni(110). The vibronic progressions revealed by means of the resonant tunneling mechanism (see picture; EF=Fermi level) were compared with those probed by tunneling-induced fluorescence.

    28. Optical Characteristics of Atomic Force Microscopy Tips for Single-Molecule Fluorescence Applications (pages 976–983)

      Alexander Gaiduk, Ralf Kühnemuth, Matthew Antonik and Claus A. M. Seidel

      Version of Record online: 6 APR 2005 | DOI: 10.1002/cphc.200400485

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      Testing tips: Several commercial atomic force microscopy (AFM) cantilevers are shown to have light scattering and luminescence characteristics suitable for combined single-molecule AFM/fluorescence experiments. For most of the cantilevers tested, the signal is comparable to the scattering signal from the surrounding buffer when the focus of the optical setup (see figure) is more than 300 nm away from the tip. The cantilevers were investigated with a diffraction-limited scanning confocal fluorescence microscopy setup.

    29. Two-Hybrid Fluorescence Cross-Correlation Spectroscopy Detects Protein–Protein Interactions In Vivo (pages 984–990)

      Nina Baudendistel, Gabriele Müller, Waldemar Waldeck, Peter Angel and Jörg Langowski

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200400639

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      Protein–protein interactions have been observed in vivo using fluorescence cross-correlation spectroscopy (FCCS). The authors report the FCCS detection of the interactions between two autofluorescent proteins in living HeLa cells (see figure), using fusion proteins between Fos and the enhanced green fluorescent protein (EGFP), as well as Jun and the monomeric red fluorescent protein 1 (mRFP1).

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      Preview: ChemPhysChem 5/2005 (page 998)

      Version of Record online: 22 APR 2005 | DOI: 10.1002/cphc.200590008