Content
2008
Photophysics of New Water-Soluble Terrylenediimide Derivatives and Applications in Biology
15.12.2008
The photophysical properties of three new water-soluble terrylenediimide (WS-TDI) derivatives are investigated and their utilization in biological experiments is demonstrated. Each of these dyes can be excited in the far red region of the visible spectrum, making them good candidates for in-vivo studies. Single-molecule techniques characterize their photophysics that is, the number of emitted photons, blinking characteristics and survival times until photobleaching takes place. All three dyes exhibit bright fluorescence, as well as an extremely high resistance against photodegradation compared to other well-known fluorophores. Due to their different characteristics the three new WSTDI derivatives are suitable for specialized biological applications. WS-TDI dodecyl forms non-fluorescent aggregates in water which can be disrupted in a hydrophobic environment leading to a monomeric fluorescent form. Due to its high lipophilicity WS-TDI dodecyl anchors efficiently in lipid bilayers with its alkyl chain and hence can be ideally used to image membranes and membrane-containing compartments in living cells. In contrast, the positively charged WS-TDI pyridoxy is a new type of chromophore in the WS-TDI family. It is fully solubilized in water forming fluorescent monomers and is successfully used to label the envelope of herpes simplex viruses. Finally, it is shown that a WS-TDI derivative functionalized with N-hydroxysuccinimide ester moiety (WS-TDI/NHS ester) provides a versatile reactive dye molecule for the specific labelling of amino groups in biomolecules such as DNA.
Accelerated and Efficient Photochemistry from Higher Excited Electronic States in Fulgide Molecules
02.12.2008
The Journal of Physical Chemistry,
2008,
112, 51,
13364 - 71
published on 02.12.2008
The Journal of Physical Chemistry , online article
The Journal of Physical Chemistry , online article
The photoinduced electrocyclic ring-opening of a fluorinated indolylfulgide is investigated by stationary and ultrafast spectroscopy in the UV/vis spectral range. Photoreactions, initiated by optical excitation into the S1 (570 nm) and SN (340 nm) absorption band of the closed isomer, lead to considerable differences in reaction dynamics and quantum yields. Transient absorption studies point to different reaction pathways depending on the specific excitation wavelength: excitation into the S1 state leads to the known reaction behavior with a picosecond decay to the ground state and a small quantum yield of 7% for the photoproduct. The SN state shows an unexpected long lifetime of 0.5 ps. The photoreaction starting from the SN state leads to a large extent directly to the product ground state and back to the educt ground state. This results in an increased reaction quantum yield of 28%. In contradiction to Kasha’s rule, the S1 state is only populated with an efficiency of 38%. The observed behavior strongly differs from the expected picture with fast relaxation into the S1 state and a subsequent ring-opening reaction starting from the lowest excited electronic state. Quantum chemical calculations confirm and complement the experimental findings allowing a sound molecular interpretation to be obtained.
Design criteria for optimal photosynthetic energy conversion
31.10.2008
Chemical Physics Letters,
2008,
446, 4-6,
209- 13
published on 31.10.2008
Chemical Physics Letters , online article
Chemical Physics Letters , online article
Photochemical solar energy conversion is considered as an alternative of clean energy. For future light converting nano-machines photosynthetic reaction centers are used as prototypes optimized during evolution. We introduce a reaction scheme for global optimization and simulate the ultrafast charge separation in photochemical energy conversion. Multiple molecular charge carriers are involved in this process and are linked by Marcus-type electron transfer. In combination with evolutionary algorithms, we unravel the biological strategies for high quantum efficiency in photosynthetic reaction centers and extend these concepts to the design of artificial photochemical devices for energy conversion.
A nano-positioning system for macromolecular structural analysis
12.10.2008
Nature Methods,
2008,
DOI:10.1038/NMETH.1259,
published on 12.10.2008
Nature Methods, online article
Nature Methods, online article
Very often, the positions of flexible domains within macromolecules as well as within macromolecular complexes
cannot be determined by standard structural biology methods.
To overcome this problem, we developed a method that uses
probabilistic data analysis to combine single-molecule measurements with X-ray crystallography data. The method
determines not only the most likely position of a fluorescent
dye molecule attached to the domain but also the complete
three-dimensional probability distribution depicting the
experimental uncertainty. With this approach, single-pair
fluorescence resonance energy transfer measurements can
now be used as a quantitative tool for investigating the position
and dynamics of flexible domains within macromolecular
complexes. We applied this method to find the position of
the 5¢ end of the nascent RNA exiting transcription elongation
complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.
Nanoparticle Self-Assembly on a DNA-Scaffold Written by Single-Molecule Cut-and-Paste
01.10.2008
Self-assembly guided by molecular recognition has in the past been employed to assemble nanoparticle superstructures like hypercrystals or nanoparticle molecules. An alternative approach, the direct molecule-by-molecule assembly of nanoscale superstructures, was demonstrated recently. Here we present a hybrid approach where we first assemble a pattern of binding sites one-by-one at a surface and then allow different nanoparticles to attach by self-assembly. For this approach, biotin bearing DNA oligomers were picked up from a depot using a cDNA strand bound to an AFM tip. These units were deposited in the target area by hybridization, forming a recognition pattern on this surface. Fluorescent semiconductor nanoparticles conjugated with streptavidin were allowed to assemble on this scaffold and to form the final nanoparticle superstructures.
live nanoparticle self-assembly movie
Mechanoenzymatics of titin kinase
03.09.2008
PNAS,
2008,
Vol. 105 no. 36 (www.pnas.org/cgi/doi/10.1073/pnas.0805034105),
13385-90
published on 09.09.2008
www.pnas.org, online article
www.pnas.org, online article
Biological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during
strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation
and substrate turnover.
Dynamics of photoinduced endosomal release of polyplexes
07.06.2008
Journal of Controlled Release,
2008,
doi:10.1016/j.jconrel.2008.06.001,
published on 07.06.2008
Journal of Controlled Release, online article
Journal of Controlled Release, online article
Endosomal escape is a well-known bottleneck for successful delivery of macromolecular drugs and genes. Photochemical disruption of endosomal membranes is an approach to overcome this bottleneck. In this study, we used the photosensitizer disulphonated meso-tetraphenylporphine with sulfonate groups on adjacent phenyl rings (TPPS2a) to investigate photoinduced endosomal release in living cells with high resolution fluorescence wide-field microscopy in real time. We studied the release dynamics of 10 kDa dextran and polyplexes consisting of DNA condensed with the cationic polymers linear polyethyleneimine (LPEI), poly-(L)-lysine (PLL) or poly-(D)-lysine (PDL). By means of dual-color microscopy and the use of double-labeled polyplexes DNA and polymer were imaged simultaneously. We show that the characteristics of the cationic polymer significantly influence the release behavior of the polyplexes. The release of dextran occurred within 100 ms. For LPEI/DNA particles, LPEI quickly spread throughout the cytosol similar to dextran, whereas DNA was released slowly (within 4 s) and remained immobile thereafter. In case of PLL particles, both DNA and polymer showed quick release. PDL particles remained condensed upon photosensitizer activation. In addition, we demonstrate that TPPS2a has biological side effects. Besides stop of microtubule dynamics in the dark, the movement of endosomes ceased after photosensitizer activation.
Simultaneous Transport of Different Localized mRNA Species Revealed by Live-Cell Imaging
09.05.2008
Traffic,
2008,
9 (doi:10.1111/j.1600-0854.2008.00763.x),
1256
published on 09.05.2008
Traffic, online article
Traffic, online article
Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, differentmRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 differentmRNAs to the bud with the help of theMyo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the
RNAtag sequences.Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the
transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles andcotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.
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Monitoring Protein Conformation along the Pathway of Chaperonin-Assisted Folding
04.04.2008
The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70).Here we employed single molecule and ensemble FRET to monitor the conformational transitions of amodel substrate as
it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding,with a fraction ofmolecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding andrapidmobilizationofprotein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES,
completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic
collapse.
Chemical control of Hemithioindigo-photoisomerization – Substituent-effects on different molecular parts
02.03.2008
Chemical Physics Letters,
2008,
455,
197–201
published on 02.03.2008
Chemical Physics Letters, online article
Chemical Physics Letters, online article
Hemithioindigo-molecules show a photochromic behavior due to a photo-induced Z/E-isomerization on a picosecond timescale. Changes in the reaction kinetics, caused by polar substituents attached to the thioindigo-moiety, are studied using time-resolved absorption spectroscopy. The experiments reveal that substituents in the thioindigo-part influence the reaction dynamics in a different manner as previously found for stilbene-substitution. The results are discussed in the context of inductive- and resonanceeffects and lead to an improved understanding of the molecular mechanisms of the Hemithioindigophotoreaction.
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PIE: eine multidimensionale Fluoreszenzmethode
01.03.2008
Bei PIE werden gepulste Anregungsquellen verwendet, wodurch die Information zugänglich ist, welche von ihnen für die Emission eines detektierten Photons verantwortlich
war. Diese experimentell leicht zu installierende
Technik bietet zahlreiche Vorteile, vor allem bei Untersuchungen an mehrfarbig markierten Molekülkomplexen. Beispielsweise besteht mit PIE die Möglichkeit, das
spektrale Übersprechen in abbildenden Techniken
(z. B. Laser Scanning- oder Weitfeld- Mikroskopie) oder bei der Fluoreszenz-Korrelations-Spektroskopie zu eliminieren. Ferner
können fehlerfreie Förster-Resonanz-Energie-Transfer-Messungen (FRET) durchgeführt sowie die Stöchiometrie von Molekülkomplexen in Einzelmolekül-Experimenten bestimmt
werden.
Hydrophobic and Hofmeister Effects on the Adhesion of Spider Silk Proteins onto Solid Substrates: An AFM-Based Single-Molecule Study
19.02.2008
AFM-based single-molecule force spectroscopy has been used to study the effect of Hofmeister salts and protein hydrophobicity on the adhesion of recombinant spider silk proteins onto solid substrates. Therefore, a molecular probe consisting of a spider silk protein and an AFM tip has been developed, which (i) is a well-defined, small system that can be simulated by molecular dynamics simulations, (ii) allows access to the whole soluble concentration range for ions, and (iii) provides the distribution of desorption forces rather than just ensemble-averaged mean values. The measured desorption forces follow the Hofmeister series for anions (H2PO4-, Cl-, I-) with a stabilizing energy of more than 15 kBT for 5 M NaH2PO4. Moreover, this effect is influenced by the hydrophobicity of the spider silk protein, indicating that hydrophobic and Hofmeister effects are closely related.
Conformational changes of a Swi2/Snf2 ATPase during its mechano-chemical cycle
11.02.2008
Nucleic Acids Research,
2008,
doi: 10.1093/nar/gkn040,
published on 11.02.2008
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Remodelling protein nucleic acid interfaces is an important biological task, which is often carried out by nucleic acid stimulated ATPases of the Swi2/Snf2 superfamily. Here we study the mechano-chemical cycle of such an ATPase, namely the catalytic
domain of the Sulfolobus solfataricus Rad54 homologue (SsoRad54cd), by means of fluorescence resonance energy transfer (FRET). The results of the FRET studies show that the enzyme can be found in (at least) two different possible conformations in solution. An open conformation, consistent with a recently reported crystal structure, is converted into a closed conformation after DNA
binding. Upon subsequent binding of ATP no further change in conformation can be detected by the FRET measurements. Instead, a FRET detectable conformational change occurs after ATP hydrolysis and prior to ADP release, suggesting a powerstroke that is linked to phosphate release. Based on these data we will present a new model for the mechanochemical cycle of this enzyme. This scheme in turn provides a working model for understanding the function of other members of the Swi2/Snf2 family.
Single Molecule Fluorescence of Native and Refolded Peridinin–Chlorophyll–Protein Complexes
17.01.2008
Journal of Fluorescence,
2008,
18,
611-7
published on 17.01.2008
Journal of Fluorescence, online article
Journal of Fluorescence, online article
Single molecule spectroscopy was applied to study the optical properties of native and refolded peridinin–chlorophyll–protein (PCP) complexes. The native system is a trimer with six chlorophyll a (Chl a) molecules, while the refolded one contains two Chl a and resembles
structurally and spectroscopically the PCP monomer. The fluorescence emission of single PCP complexes strongly broadens with increasing excitation power. Simultaneously, the distribution of fluorescence maximum frequencies is also broadened. These spectral changes are attributed to photoinduced conformational changes of the protein that influence the fluorescence of embedded chromophores. Comparison of fluorescence intensities measured for PCP complexes in two different solvents indicates that the native
PCP trimers are preserved in EDTA Tris buffer, while in PVA polymer matrix only monomers are stable.
Single-molecule tracking of mRNA exiting from RNA polymerase II
08.01.2008
Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5´ end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active center cleft via the previously proposed exit tunnel and then disengages from the enzyme surface. When the RNA reaches lengths of 26 and 29 nt, its 5´ end associates with Pol II at the base of the dock domain. Because the initiation factor TFIIB binds to the dock domain and exit tunnel, exiting RNA may prevent TFIIB reassociation during elongation. RNA further extends toward the linker connecting to the polymerase C-terminal repeat domain (CTD), which binds the 5´-capping enzyme and other RNA processing factors.
The Hammett Relationship and Reactions in the Excited Electronic State Hemithioindigo Z/E-Photoisomerization
05.01.2008
The Journal of Physical Chemistry,
2008,
112, 4,
581- 88
published on 05.01.2008
The Journal of Physical Chemistry , online article
The Journal of Physical Chemistry , online article
The photochemical reaction dynamics of a set of photochromic compounds based on thioindigo and stilbene molecular parts (hemithioindigos, HTI) are presented. Photochemical Z/E isomerization around the central double bond occurs with time constants of 216 ps (Z → E) and 10 ps (E → Z) for a 5-methyl-hemithioindigo. Chemical substitution on the stilbene moiety causes unusually strong changes in the reaction rate. Electron-donating substituents in the position para to the central double bond (e.g., para-methoxy) strongly accelerate the reaction, while the reaction is drastically slowed by electron-withdrawing groups in this position (e.g., para-nitrile). We correlate the experimental data of seven HTI-compounds in a quantitative manner using the Hammett equation and present a qualitative explanation for the application of ground-state Hammett constants to describe the photoisomerization reaction.
