Content
2009
Polymer Adhesion at the Solid–Liquid Interface Probed by a Single–Molecule Force Sensor
01.12.2009
A method based on atomic force microscopy is used to delineate the properties that determine single-molecule adhesion onto solid substrates in aqueous environment. Hydrophobicity as well as electrical properties of the substrate and the polymer are varied. In addition, the influence of the solvent composition, in particular the effect of ions, on the molecular adhesion at the solid–liquid interface is studied. Surprisingly, the polymer and surface-related properties account for only small changes in adhesion force, while dissolved ions show a much larger effect. These results point towards the energy of solvation as the most important contribution to adhesion for a wide variety of polymers and substrate materials.
The detailed balance limit of photochemical energy conversion
16.11.2009
Phys. Chem. Chem. Phys.,
2009,
DOI: 10.1039/B914552D,
12, 422-432
published on 16.11.2009
Phys. Chem. Chem. Phys., online article
Phys. Chem. Chem. Phys., online article
Limits and optimization of a solar energy conversion system consisting of a photochemical charge separating unit coupled to an energy storage state are explored by multi-objective genetic
algorithms. Pareto fronts were evaluated to obtain information about the ideal parameter combinations, guaranteeing highest efficiency. The light absorbing and charge separating unit is
described by a chain of chromophores and electron carriers, connected by Marcus type electron transfer processes. It is coupled to the thermal equilibrium of charge conduction and transport in an energy storage system according to the principle of detailed balance. In addition to our previous findings for an optimal charge separation unit, consisting of a minimum number of charge carriers with adapted recombination and reaction rates, the complete photochemical unit must fulfil further requirements. Low reorganization energies are found to be essential for the initial charge separation steps and can be realized by a low dielectric constant in the local environment. The identified optimal operation rates can be realized by antenna systems adapted to the illumination conditions. For standard solar illumination and a realistic parameter setting energy conversion efficiencies up to 26.8% are predicted, comparable to the limit (31.8%) of ideal single junction semiconductor solar cells.
Single Molecule Adhesion Mechanics on Rough Surfaces
11.11.2009
The nanostructure of materials is supposed to affect polymer adhesion at the solid/liquid interface by variations of the exposed surface area. In contrast, our studies on the nanotribology of single recombinant spider silk proteins onto smooth and rough surfaces made of surgical stainless steel show no effect of the surface morphology. This is explained by the velocity dependence of the desorption process, which reveals a negligible friction and a high in-plane mobility at the molecular scale. We compare our results to all atomistic molecular dynamics simulations that allow estimating an upper limit for the molecular friction coefficient.
Visualizing Single-Molecule Diffusion in Nanochannel Systems
10.11.2009
in: Single Particle Tracking and Single Molecule Energy Transfer, Eds.: C. Bräuchle, D. C. Lamb, J. Michaelis,
Wiley VCH Weinheim (2009),
p. 309, ISBN: 978-3-527-32296-1
published on 10.11.2009
Wiley homepage
Wiley homepage
Closing a gap in the literature, this handbook gathers all the information on single particle tracking and single molecule energy transfer. It covers all aspects of this hot and modern topic, from detecting virus entry to membrane diffusion, and from protein folding using spFRET to coupled dye systems, as well recent achievements in the field. Throughout, the first-class editors and top international authors present content of the highest quality, making this a must-have for physical chemists, spectroscopists, molecular physicists and biochemists.
The titin-telethonin complex is a directed, superstable molecular bond in the muscle Z-disk
21.07.2009
PNAS,
2009,
doi: 10.1073/pnas.0902312106,
vol. 106 no. 32 13307-133310 ,
published on 09.11.2009
PNAS, online article
PNAS, online article
Mechanical stability of bonds and protein interactions has recently
become accessible through single molecule mechanical experiments. So far, mechanical information about molecular bond
mechanics has been largely limited to a single direction of force
application. However, mechanical force acts as a vector in space
and hence mechanical stability should depend on the direction of
force application. In skeletal muscle, the giant protein titin is anchored in the Z-disk by telethonin. Much of the structural integrity of the Z-disk hinges upon the titin-telethonin bond. In this paper we show that the complex between the muscle proteins titin and telethonin forms a highly directed molecular bond. It is designed to resist ultra-high forces if they are applied in the direction along which it is loaded under physiological conditions, while it breaks easily along other directions. Highly directed molecular bonds match in an ideal way the requirements of tissues subject to mechanical stress.
Adsorption Mechanism of Polypeptides and Their Location at Hydrophobic Interfaces
09.11.2009
Chem Phys Chem,
2009,
doi:10.1002/cphc.200900574,
published on 09.11.2009
Chem. Phys. Chem., online article
Chem. Phys. Chem., online article
The adhesion of polypeptides and proteins at interfaces is important for a wide variety of systems—from the adhesion onto vessels during their production process over functional coatings to protein–membrane interactions in single cells. Herein, we apply an atomic-force-microscopy-based single-molecule method and poly-D-tyrosine to determine the adhesion strength and location of polypeptides at interfaces. Surprisingly, the support (solid, liquid or gas) hardly influences the adhesion in an aqueous environment, while the addition of ethanol to the solvent cuts the adhesion in half. These findings allow us to propose that the adsorbed polypeptide spans over both the depletion layer and the hydrophobic hydration layer to facilitate a compensation mechanism between dispersive and hydration forces.
Dynamics of HIV-1 Assembly and Release
06.11.2009
PLoS Pathog.,
2009,
5(11),
e1000652, doi:10.1371/journal.ppat.1000652
published on 06.11.2009
PLOS Pathog., online article
PLOS Pathog., online article
Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ~5x10^-3 per second, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ~1,500 +/- 700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.
DNA based molecular motors
03.10.2009
Physics of Life Reviews,
2008,
6,
250–266, doi:10.1016/j.plrev.2009.09.001
published on 03.10.2009
Physics of Life Review, online article
Physics of Life Review, online article
Most of the essential cellular processes such as polymerisation reactions, gene expression and regulation are governed by mechanical processes. Controlled mechanical investigations of these processes are therefore required in order to take our understanding of molecular biology to the next level. Single-molecule manipulation and force spectroscopy have over the last 15 years been developed into extremely powerful techniques. Applying these techniques to the investigation of proteins and DNA molecules has led to a mechanistic understanding of protein function on the level of single molecules. As examples for DNA based molecular machines we will describe single-molecule experiments on RNA polymerases as well as on the packaging of DNA into a viral capsid—a process that is driven by one of the most powerful molecular motors.
YEAH! CIPSM-Professor Jens Michaleis receives big €-EU-Grant
01.10.2009
2009,
published on 01.10.2009
ERC
ERC
CIPSM-Investigator Jens Michaelis has done it! Jens received a grant of nearly 1,5 million Euros from the European Research Council for the next five years. Jens will explore the understanding of the molecular mechanism of nucleosome remodelling using single-molecule fluorescence resonance energy transfer (FRET).
In eukaryotic cells the DNA is packaged into nucleosomes and higher order structures which lead to a condensation and protection of the DNA. During important cellular processes such as transcription or replication access to the DNA has to be granted. This is facilitated by ATP dependent nucleosome remodelling. The mechanistic details how the involved remodelling complexes succeed in providing access to the nucleosomal DNA are currently in spite of large experimental efforts not well understood. The aim of the proposal is to unravel the molecular mechanism of nucleosome remodelling using single-molecule fluorescence resonance energy transfer (FRET). By putting labels on the nucleosomal DNA, the histones or the remodellers we will - step by step - determine the conformational changes that occur during remodelling and use this information to build a mechanistic model. We will also use the controlled assembly of 30 nm fibers from purified components in order to determine how remodelling occurs in this structurally restricted environment. The large size of the chromatin fibers will dictate that in addition to FRET measurements, which will again be used to investigate local motion, super-resolution microscopy need to be employed to obtain information about long-distance movements.
To this end we will use stochastic optical reconstruction microscopy (STORM), which allows for accuracy below 10 nm, thus ideally complementing the FRET approach.
In summary our experiments will lead us to a mechanistic understanding of ATP dependent nucleosome remodelling, both on mononucleosomes as well as in higher order structures. This knowledge will in return stimulate new initiatives aimed at understanding the nature and regulation of chromatin dynamics in vivo.
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A 75 MHz Light Source for Femtosecond Stimulated Raman Microscopy
30.09.2009
Optics Express,
2009,
http://dx.doi.org/10.1364/OE.17.018612,
Vol. 17, Issue 21, pp. 18612-18620
published on 30.09.2009
Optics Express, online article
Optics Express, online article
In femtosecond stimulated Raman microscopy (FSRM) a spectrally broad pulse (Raman probe) and a spectrally narrow pulse (Raman pump) interact in a sample and thereby generate a Raman spectrum of the focal volume. Here a novel light source for FSRM is presented. It consists of an 8-fs laser (repetition rate of 75 MHz) operating as Raman probe. A Yb3+ based fiber amplifier generates the Raman pump light at 980 nm. The amplifier is seeded by the spectral wing of the 8-fs laser output which ensures synchronisation of pump and probe pulses. Spectral and temporal characteristics of these pulses are reported and simultaneous recording of broadband Raman spectra relying on these pulses is demonstrated.
Disturbing interference patterns in femtosecond stimulated Raman microscopy
30.09.2009
Journal of Raman Spectroscopy,
2009,
DOI: 10.1002/jrs.2488,
Volume 41, Issue 6, pages 609–613
published on 30.09.2009
Journal of Raman Spectroscopy, online article
Journal of Raman Spectroscopy, online article
Femtosecond stimulated Raman microscopy (FSRM) is an upcoming technique in nonlinear microscopy which facilitates rapid chemical mapping. It employs femtosecond white-light pulses as probe pulses and intense picosecond pulses as pump pulses. Stimulated Raman scattering (SRS) occurs at the focus of a scanning microscope. Chemical constituents in the sample are identified via their Raman signatures. In this article, disturbing interference patterns in FSRM are reported. They are caused by a broadening of the pump pulse due to nonlinear interactions in the focal region of the microscope and reduce the signal-to-noise ratio. The properties of these modulations are explored, and the methods to suppress them are presented
Photochemistry with thermal versus optical excess energy: Ultrafast cycloreversion of indolylfulgides and indolylfulgimides
25.09.2009
Journal of Photochemistry and Photobiology A: Chemistry,
2009,
doi:10.1016/j.jphotochem.2009.07.012,
Volume 207, Issues 2–3, Pages 209–216
published on 25.09.2009
Journal of Photochemistry and Photobiology A: Chemistry, online article
Journal of Photochemistry and Photobiology A: Chemistry, online article
The dependence of the pericyclic ring-opening reaction of indolylfulgides and indolylfulgimides on excess energy is investigated by quantum efficiency measurements and by ultrafast spectroscopy. The ring-opening reaction shows a pronounced improvement of reaction efficiency up to a factor of 6, when excess energy is available either by increasing the temperature or by exciting the molecules above the 0–0-transition. Ultrafast spectroscopy allows to deduce time constants for the ring-opening reaction and leads to a theoretical model, where the redistribution of excess energy among different vibrational modes is considered. The analysis shows that excess energy supplied by optical excitation accelerates the ring-opening reaction less efficiently than thermal energy. Apparently vibrational relaxation from highly excited modes to modes promoting the ring-opening reaction is not completed within the ∼10 ps duration of the ring-opening reaction.
Real-time Nanomicroscopy via Three-Dimensional Single-Particle Tracking
16.09.2009
We developed a new method for real-time, three-dimensional tracking of fluorescent particles. The instrument is based on a laser-scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resolution in the lateral dimensions and 50 nm in the axial dimension at a temporal resolution of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual-color, wide-field setup. Dual-color fluorescence wide-field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent-labeled artificial viruses in tubulin-eGFP expressing HUH7 cells. The resulting trajectories can be used to investigate the microtubule network with super resolution.
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Single-molecule force spectroscopy distinguishes target binding modes of calmodulin
10.08.2009
PNAS,
2009,
doi: 10.1073/pnas.0904654106,
vol. 106 no. 34 14361-14366 ,
published on 10.08.2009
PNAS, online article
PNAS, online article
The eukaryotic signaling protein calmodulin (CaM) can bind to
more than 300 known target proteins to regulate numerous
functions in our body in a calcium-dependent manner. How CaM
distinguishes between these various targets is still largely unknown. Here, we investigate fluctuations of the complex formation of CaM and its target peptide sequences using single-molecule force spectroscopy by AFM. By applying mechanical force, we can steer a single CaM molecule through its folding energy landscape from the fully unfolded state to the native target-bound state revealing equilibrium fluctuations between numerous intermediate states. We find that the prototypical CaM target sequence skMLCK, a fragment from skeletal muscle myosin light chain kinase, binds to CaM in a highly cooperative way, while only a lower degree of interdomain binding cooperativity emerges for CaMKK, a target peptide from CaM-dependent kinase kinase. We identify minimal binding motifs for both of these peptides, confirming that affinities of target peptides are not exclusively determined by their pattern of hydrophobic anchor residues. Our results reveal an association mode for CaMKK in which the peptide binds strongly to only partially Ca2plus-saturated CaM. This binding mode might allow for a fine-tuning of the intracellular response to changes in Ca2plus concentration.
Folding DNA into Twisted and Curved Nanoscale Shapes
07.08.2009
Science,
2009,
325 (DOI: 10.1126/science.1174251),
725
published on 07.08.2009
Science, online article
Science, online article
We demonstrate the ability to engineer complex shapes that twist and curve at the nanoscale from DNA. Through programmable self-assembly, strands of DNA are directed to form a custom-shaped bundle of tightly cross-linked double helices, arrayed in parallel to their helical axes. Targeted
insertions and deletions of base pairs cause the DNA bundles to develop twist of either handedness or to curve. The degree of curvature could be quantitatively controlled, and a radius of curvature as tight as 6 nanometers was achieved. We also combined multiple curved elements to build several different types of intricate nanostructures, such as a wireframe beach ball or square-toothed gears.
Perspective "Designer Curvature"
Stability and reaction dynamics of trifluorinated indolylfulgides
06.08.2009
Chemical Physics Letters,
2009,
doi:10.1016/j.cplett.2009.07.013,
Volume 477, Issues 4–6, Pages 298–303
published on 06.08.2009
Chemical Physics Letters, online article
Chemical Physics Letters, online article
Quantum efficiencies and ultrafast dynamics of the ring-closure and ring-opening reaction of a trifluorinated dicyclopropyl indolylfulgide with improved photostability are investigated by stationary and ultrafast absorption spectroscopy. The ring-closure reaction occurs on the time scale of 200 fs and is found to be temperature independent (T = 287–333 K). However, an activated behaviour is observed for the ring-opening reaction. A comparison with the corresponding non-substituted indolylfulgide shows that the dicyclopropyl group favours the open isomer via lower cyclisation and higher cycloreversion quantum efficiencies and faster dynamics of the ring-opening reaction.
Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex
20.07.2009
Nucleic Acids Research,
2009,
doi:10.1093/nar/gkp601,
published on 20.07.2009
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Crystallographic studies of the RNA polymerase II
(Pol II) elongation complex (EC) revealed the
locations of downstream DNA and the DNA-RNA
hybrid, but not the course of the nontemplate
DNA strand in the transcription bubble and the
upstream DNA duplex. Here we used singlemolecule
Fluorescence Resonance Energy Transfer (smFRET) experiments to locate nontemplate and upstream DNA with our recently developed Nano Positioning System (NPS). In the resulting complete model of the Pol II EC, separation of the nontemplate from the template strand at position +2 involves interaction with fork loop 2. The
nontemplate strand passes loop b10-b11 on the Pol
II lobe, and then turns to the other side of the cleft
above the rudder. The upstream DNA duplex exits at
an approximately right angle from the incoming
downstream DNA, and emanates from the cleft
between the protrusion and clamp. Comparison
with published data suggests that the architecture
of the complete EC is conserved from bacteria to
eukaryotes and that upstream DNA is relocated
during the initiation–elongation transition.
Influence of the Charge at D85 on the Initial Steps in the Photocycle of Bacteriorhodopsin
08.07.2009
The Journal of Physical Chemistry,
2009,
97,
267- 76
published on 08.07.2009
Biophysical Journal , online article
Biophysical Journal , online article
Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.
Nanostructured Silica Materials As Drug-Delivery Systems for Doxorubicin: Single Molecule and Cellular Studies
02.07.2009
Nano Lett.,
2009,
9, 8 (DOI: 10.1021/nl9011112),
2877
published on 02.07.2009
Nano Letters, online article
Nano Letters, online article
We apply mesoporous thin silica films with nanometer-sized pores as drug carriers and incorporate the widely used anticancer drug Doxorubicin. Through single-molecule based measurements, we gain mechanistic insights into the drug diffusion inside the mesoporous film, which governs the drug-delivery at the target-site. Drug dynamics inside the nanopores is controlled by pore size and surface modification. The release kinetics is determined and live-cell measurements prove the applicability of the system for drug-delivery. This study demonstrates that mesoporous silica nanomaterials can provide solutions for current challenges in nanomedicine.
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Single molecule force measurements delineate salt, pH and surface effects on biopolymer adhesion
01.06.2009
Physical Biology,
2009,
doi:10.1088/1478-3975/6/2/025004,
published on 01.06.2009
Phys. Biol., online article
Phys. Biol., online article
In this paper we probe the influence of surface properties, pH and salt on the adhesion of recombinant spider silk proteins onto solid substrates with single molecule force spectroscopy. A single engineered spider silk protein (monomeric C16 or dimeric (QAQ)8NR3) is covalently bound with one end to an AFM tip, which assures long-time measurements for hours with one and the same protein. The tip with the protein is brought into contact with various substrates at various buffer conditions and then retracted to desorb the protein. We observe a linear dependence of the adhesion force on the concentration of three selected salts (NaCl, NaH2PO4 and NaI) and a Hofmeister series both for anions and cations. As expected, the more hydrophobic C16 shows a higher adhesion force than (QAQ)8NR3, and the adhesion force rises with the hydrophobicity of the substrate. Unexpected is the magnitude of the dependences—we never observe a change of more than 30%, suggesting a surprisingly well-regulated balance between dispersive forces, water-structure-induced forces as well as co-solute-induced forces in biopolymer adhesion.
Dynamics of magnetic lipoplexes studied by single particle tracking in living cells
07.04.2009
Journal of Controlled Release,
2009,
137,
136-45
published on 07.04.2009
Journal of Controlled Release, online article
Journal of Controlled Release, online article
Magnetofection, gene delivery under the influence of a magnetic field, is a technique to increase transfection efficiency by enforcing gene vector contact with a target cell. Mechanisms of magnetic lipoplex internalization and intracellular details of magnetofection are still unknown. In this study, cellular dynamics
of magnetic lipoplexes were examined in real time by means of highly sensitive dual-color fluorescence microscopy. Single particle tracking of magnetic lipoplexes provided trajectories representing the movement of the lipoplexes during internalization and subsequent intracellular processes. Magnetic lipoplexes show a three-phase behavior similar to polyplexes. During phase I lipoplexes are attached to the cell surface and
show slow cooperative transport behavior. Phase II takes place inside the cell and was characterized by anomalous and confined diffusion. Phase III represented active transport along microtubules inside the cell. The majority of lipoplexes were internalized via endocytosis during phase I. On later time scales the formation of a perinuclear ring was observed. Persisting colocalization of fluid phase marker and lipoplexes after 24 h indicated slow endosomal release. In short, the internalization characteristics of magnetic lipoplexes are very similar to that of polyplexes. Furthermore our results suggest that the magnetic field induces an increased concentration of magnetic complexes on the cell surface resulting in higher transfection efficiency.
Atomic force microscopy spring constant determination in viscous liquids
31.03.2009
Review of Scientific Instruments,
2009,
doi:10.1063/1.3100258,
published on 31.03.2009
Rev. Sci. Instrum., online article
Rev. Sci. Instrum., online article
The spring constant of cantilever in atomic force microscopy (AFM) is often calibrated from thermal noise spectra. Essential for accurate implementation of this “thermal noise method” is an appropriate fitting function and procedure. Here, we survey the commonly used fitting functions and examine their applicability in a range of environments. We find that viscous liquid environments are extremely problematic due to the frequency dependent nature of the damping coefficient. The deviations from the true spring constant were sometimes more than 100% when utilizing the fit routines built into the three investigated commercial AFM instruments; similar problems can arise with homebuilt AFMs. We discuss the reasons for this problem, especially the limits of the fitting process. Finally, we present a thermal noise based procedure and an improved fit function to determine the spring constant with AFMs in fluids of various viscosities.
Thymine Dimerization in DNA Model Systems: Cyclobutane Photolesion Is Predominantly Formed via the Singlet Channel
23.03.2009
Photoreactions induced by ultraviolet radiation are among the
most important external hazards for the integrity of DNA.1 The
photolesion with the highest abundance is the [2 + 2] photoaddition
of thymine bases adjacent on a DNA strand.2 This photoaddition
yields a cyclobutane pyrimidine dimer (CPD). The formation of
the CPD lesion has first been described in 1960.3,4 Nevertheless,
the kinetics of its formation is still under debate. In solution the
photodimerization of single thymine bases involves a triplet state.5,6
A reaction via a singlet channel is precluded in this diffusion limited
process since singlet excitations in thymine are too short-lived.7,8
In DNA strands thymine bases are kept in proximity by the
sugar-phosphate backbone and the diffusion limit does not apply.
So in DNA the CPD lesion could in principle be formed via excited
singlet or triplet states.
Single-Molecule Investigations of a Photoswitchable Nanodevice
04.03.2009
Small,
2009,
DOI: 10.1002/smll.200801549,
Volume 5, Issue 10, pages 1169–1175,
published on 04.03.2009
Small, online article
Small, online article
Due to the specificity of Watson–Crick base pairing, DNA is an excellent molecule for the fabrication of nanostructures. It has been shown that DNA can be used as a scaffold for positioning proteins and synthetic molecules with nanometer accuracy. As the next step in adding complexity and functionality to these nanodevices, optical addressability is incorporated. The fluorescent protein Dronpa, which can be optically switched between a fluorescent state and a dark state, is mounted on a DNA scaffold in the proximity of a synthetic fluorophore. Hence, the system can be optically switched between the dark state and an optically active state that undergoes Förster resonance energy transfer. As nanodevices operate as individual units, the functionality of the device is analyzed using single-molecule microscopy. The physical characteristics of nanodevices make them well suited as probes for investigating cellular processes or as shuttles for gene therapy. Hence, the functionality of the nanodevice is verified in the context of cellular measurements.
The large conformational changes of Hsp90 are only weakly coupled to ATP hydrolysis
01.03.2009
Nature Structual & Molecular Biology,
2009,
16,
281 - 86
published on 01.03.2009
www.nature.com, online article
www.nature.com, online article
The molecular chaperone heat-shock protein 90 (Hsp90) is one of the most abundant proteins in unstressed eukaryotic cells. Its function is dependent on an exceptionally slow ATPase reaction that involves large conformational changes. To observe these conformational changes and to understand their interplay with the ATPase function, we developed a single-molecule assay that allows examination of yeast Hsp90 dimers in real time under various nucleotide conditions. We detected conformational fluctuations between open and closed states on timescales much faster than the rate of ATP hydrolysis. The compiled distributions of dwell times allow us to assign all rate constants to a minimal kinetic model for the conformational changes of Hsp90 and to delineate the influence of ATP hydrolysis. Unexpectedly, in this model ATP lowers two energy barriers almost symmetrically, such that little directionality is introduced. Instead, stochastic, thermal fluctuations of Hsp90 are the dominating processes.
Mutations of the peripheral antenna complex LH2–correlations of energy transfer time with other functional properties
23.02.2009
The peripheral antenna complex (LH2) of wild type and mutants of the LH2 α-subunit of purple bacterium Rhodobacter sphaeroides are investigated by transient and stationary absorption spectroscopy. The time for energy transfer from the bacteriochlorophyll (BChl) molecules B800 to B850 is found to depend on the mutation and varies between 0.7 ps for wild type LH2 to 1.0 ps. Also quantities of functional properties (peak position, width and relative band intensity) of the B850 absorption band depend on mutation. In correlation plots connections between different quantities and the energy transfer time are identified. The observed correlations indicate that the mutations influence the binding affinities of the BChl molecules. Heterogeneity in structure and changes in the occupation of B800 and B850 rings lead to the observed variation of functional properties.
Wavelength and solvent independent photochemistry: the electrocyclic ring-closure of indolylfulgides
11.02.2009
Photochemical & Photobiological Sciences,
2009,
8,
528- 34
published on 11.02.2009
Photochemical & Photobiological Sciences , online article
Photochemical & Photobiological Sciences , online article
A wavelength and solvent dependent study of a photochromic indolylfulgide is presented. The ring-closure reaction is characterized using stationary and time-resolved spectroscopy with femtosecond time resolution. After excitation into the first excited singlet state (S1) the photoprocesses proceed on ultrafast timescales (0.3–0.45 ps) in both polar and non-polar solvents. Excitation into higher electronic states results in similar reaction kinetics as found for S1 excitation. A simple kinetic scheme can be established for the photoprocesses under all different experimental conditions: as expected from organic textbooks neither the solvent surroundings nor the excitation wavelength strongly alter the reaction scheme. The experimental study demonstrates that the ring-closure reaction of photochromic indolylfulgides can be considered as a very robust photoprocess: this fact may lead to a great variety of different applications where the reaction dynamics of the molecular switch are not disturbed by any surrounding effects.
Ligand-Dependent Equilibrium Fluctuations of Single Calmodulin Molecules
31.01.2009
Single-molecule force spectroscopy allows superb mechanical control of protein conformation. We used a custom-built low-drift atomic force microscope to observe mechanically induced conformational equilibrium fluctuations of single molecules of the eukaryotic calcium-dependent signal transducer calmodulin (CaM). From this data, the ligand dependence of the full energy landscape can be reconstructed. We find that calcium ions affect the folding kinetics of the
individual CaM domains, whereas target peptides stabilize the already folded structure. Single-molecule data of full length CaM reveal that a wasp venom peptide binds noncooperatively
to CaM with 2:1 stoichiometry, whereas a target enzyme peptide binds cooperatively with 1:1 stoichiometry. If mechanical load is applied directly to the target peptide, real-time binding/unbinding transitions can be observed.
Photochromic Bis(thiophen-3-yl)maleimides Studied with Time-Resolved Spectroscopy
14.01.2009
The Journal of Physical Chemistry,
2009,
113, 6,
1033- 39
published on 14.01.2009
The Journal of Physical Chemistry, online article
The Journal of Physical Chemistry, online article
The dynamics of the ring-closure reaction of three different bis(thiophen-3-yl)maleimides are investigated using ultrafast spectroscopy in the visible range. The structures of the molecules differ with respect to substitution of the thiophene ring and the maleimide. The experiments reveal reaction kinetics which point to the population of an excited electronic state for several nanoseconds. In the case of completely unsubstituted thiophene rings, a long excited-state lifetime (biexponential decay with 3 and 15 ns) can be observed. The remaining ultrafast absorption transients of this molecule are due to relaxational processes on the excited electronic potential energy surface. The ring-closure reaction has a small yield (<1%) and does not show up in the ultrafast absorption experiments. A dimethyl substitution of the thiophene ring results in completely different behavior: after transients related to relaxation in the excited electronic state, one finds pronounced absorption transients with τ = 16 ps which represent the partial decay of the excited electronic state and the formation of the ring-closed isomer. Another fraction of the emitting excited electronic state decays again on the few nanosecond time scale. The experiments suggest that the open isomer of the dimethyl-substituted imides exists in two conformations.
Ultrafast Hemithioindigo-based peptide-switches
04.01.2009
Chemical Physics,
2009,
113, 6358, 1-2,
103 - 10
published on 04.01.2009
Chemical Physics, online article
Chemical Physics, online article
Four newly synthesized Hemithioindigo-based peptide-switches with changing meta/para-substitution-pattern within the stilbene-part of the molecule are characterized with time-resolved absorption spectroscopy. The different substances undergo a light-induced Z/E-isomerization: the reaction proceeds on a picosecond timescale with time constants <50 ps for the Z → E photoreaction and <10 ps for the inverse process from E → Z. The reaction times strongly depend on the specific substitution-pattern: fast reaction dynamics (<10 ps) are observed for the direct attachment of electron donating groups in the para-position. The molecular mechanism of the isomerization-process can be described with the same reaction model as used for related systems. The presented Hemithioindigos are hence promising alternatives to Azobenzene-switches due to their fast switching speed, improved spectral characteristics and an improved stability.
