M. Davies, A. Wochnik, F. Feil, C. Jung, C. Bräuchle, C. Scheu, and J. Michaelis
Synchronous Emission from Nanometric Silver Particles through Plasmonic Coupling on Silver naowires
We investigated silver nanowires using correlative wide-field fluorescence and transmission electron microscopy. In the wide-field fluorescence images, synchronous emission from different distinct positions along the silver nanowires was observed. The sites of emission were separated spatially by up to several micrometers. Nanowires emitting in such cooperative manner were then also investigated with a combination of transmission electron microscopy based techniques, such as high-resolution, bright-field imaging, electron diffraction, high-angle annular dark-field imaging, and energy-dispersive X-ray spectroscopy. In particular, analyzing the chemical composition of the emissive areas using energy-dispersive X-ray spectroscopy led to the model that the active emissive centers are small silver clusters generated photochemically and that individual clusters are coupled via surface plasmons of the nanowire.
ACS Nano 6(7) (2012) 6049-6057
C. Bönisch, K. Schneider, S. Pünzeler, S.M. Wiedemann, C. Bielmeier, M. Bocola, H.C. Eberl, W. Kuegel, J. Neumann, E. Kremmer, H. Leonhardt, M. Mann, J. Michaelis, L. Schermelleh, and S.B. Hake
H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive
characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.
Nucleic Acids Research 40 (2012) 5951-5964
B. Treutlein, A. Muschielok, J. Andrecka, A. Jawhari, C. Buchen, D. Kostrewa, F. Hög, P. Cramer
and J. Michaelis
Dynamic Architecture of a Minimal RNA Polymerase II Open Promoter Complex
The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ singlemolecule Fo¨ rster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture of a minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.
Molecular Cell 46 (2012) 136-146
W. Kügel, A. Muschielok and J. Michaelis
Bayesian-Inference-Based Flourescence Correlation Spectroscopy and Single-Moledule Burst Analysis Reveal the Influence of Dye Selection on DNA Hairpin Dynamics
Fluorescence correlation spectroscopy (FCS) is a powerful tool to gain information about dynamics of biomolecules. However, the key problem is to extract the rates hidden in the FCS data by fitting the data to a meaningful model. A number of different fitting approaches have been described in recent years but the extraction of relevant information to date has still been limited by numerous experimental problems and the fact that the set of starting parameter values chosen could often predefine the result. We establish a new way to globally analyze FCS data based on Bayesian inference to overcome these issues. Moreover, the influence of other remaining experimental error sources, for example, photophysics, is excluded by additional means. Using this approach in combination with the results from single-molecule burst analysis, we investigate the kinetics of DNA hairpins labeled with a variety of different fluorescent probes as a function of the salt concentration. We find that the rates of hairpin opening and closing as well as the equilibrium constant of the transition depend on the characteristics of the dye molecules used to label the hairpin. Thus, great caution has to be used when utilizing dye molecules as reporters for the kinetics of dynamic macromolecular structures.
ChemPhysChem 13 (2012) 1013-1022
E. M. Linares, L. T. Kubota, J. Michaelis, S. Thalhammer
Enhancement of the detection limit for lateral flow immunoassays: Evaluation and comparison of bioconjugates
There is an increasing demand for convenient and accurate point-of-care tools that can detect and diagnose different stages of a disease in remote or impoverished settings. In recent years, lateral flow immunoassays (LFIA) have been indicated as a suitable medical diagnostic tool for these environments because they require little or no sample preparation, provide rapid and reliable results with no electronic components and thus can be manufactured at low costs and operated by unskilled personnel. However, even though they have been successfully applied to acute and chronic disease detection, LFIA based on gold nanoparticles, the standard marker, show serious limitations when high sensitivity is needed, such as early stage disease detection. Moreover, based on the lack of comparative information for label performance, significant optimization of the systems that are currently in use might be possible. To this end, in the presented work, we compare the detection limit between the four most used labels: colloidal-gold, silver enhanced gold, blue latex bead and carbon black nanoparticles. Preliminary results were obtained by using the biotin–streptavidin coupling as a model system and showed that carbon black had a remarkably low detection limit of 0.01 μg/mL in comparison to 0.1 μg/mL, 1 μg/mL and 1 mg/mL for silver-coated gold nanoparticles, gold nanoparticles and polystyrene beads, respectively. Therefore, as a proof of concept, carbon black was used in a detection system for Dengue fever. This was achieved by immobilizing monoclonal antibodies for the nonstructural glycoprotein (NS1) of the Dengue virus to carbon black. We found that the colorimetric detection limit of 57 ng/mL for carbon black was ten times lower than the 575 ng/mL observed for standard gold nanoparticles; which makes it sensitive enough to diagnose a patient on the first days of infection. We therefore conclude that, careful screening of detection labels should be performed as a necessary step during LFIA development in order to enhance the detection limit in a final test system.
J. of Immunological Methods 375 (2012) 264-270
F. Feil, S. Naumov, J. Michaelis, R. Valiullin, D. Enke, J. Kärger and C. Bräuchle
Single-Particle and Ensemble Diffusivities - Test of Ergodicity
(Leuchtspuren verraten Ordnung im Chaos)
Angew. Chem. Intern. Edition 51 (2012) 1152-1155
Angew. Chem. 124 (2012) 1178-1181
F. Feil, S. Naumov, J. Michaelis, R. Valiullin, D. Enke, J. Kärger, and C. Bräuchle
Single-Particle and Ensemble Diffusivities - Test of Ergodicity
Angew. Chem. 124 (2012) 1178-1181
A. Muschielok, J. Michaelis
Application of the Nano-Positioning System to the Analysis of Fluorescence Resonance Energy Transfer Networks
Single-molecule fluorescence resonance energy transfer (sm-FRET) has been recently applied to distance and position estimation in macromolecular complexes. Here, we generalize the previously published Nano-Positioning System (NPS), a probabilistic method to analyze data obtained in such experiments, which accounts for effects of restricted rotational freedom of fluorescent dyes, as well as for limited knowledge of the exact dye positions due to attachment via flexible linkers. In particular we show that global data analysis of complete FRET networks is beneficial and that the measurement of FRET anisotropies in addition to FRET efficiencies can be used to determine accurately both position and orientation of the dyes. This measurement scheme improves localization accuracy substantially, and we can show that the improvement is a consequence of the more precise information about the transition dipole moment orientation of the dyes obtained by FRET anisotropy measurements. We discuss also rigid body docking of different macromolecules by means of NPS, which can be used to study the structure of macromolecular complexes. Finally, we combine our approach with common FRET analysis methods to determine the number of states of a macromolecule.
J Phys Chem B. 2011 Oct 20;115(41):11927-37
doi: 10.1021/jp2060377Supplementary Material
D. Grohmann, J. Nagy, A. Chakraborty, D. Klose, D. Fielden, R.H. Elbright, J. Michaelis, and F. Werner
The Initiation Factor TFE and the Elongation Factor Spt4/5 Compete for the RNAP Clamp during Transcription Initiation and Elongation
TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP ‘‘clamp’’ region as a regulatory hot spot for both transcription initiation and transcription elongation.
Molecular Cell Volume 43, ISSUE 2, P263-274, July 22, 2011
B. Treutlein, J. Michaelis
Direct Observation of Single RNA Polymerase Processing through a Single Endogenous Gene in a Living Yeast Cell
Rapid advances in live-cell imaging have now enabled direct observation of the transcription of single nascent mRNA molecules from an endogenous yeast gene. A novel quantitative fluctuation analysis of fluorescently labeled mRNA revealed the kinetics of transcription initiation and the dynamics of elongation and termination (see picture; GFP=green fluorescent protein, PP7 is a bacteriophage coat protein, RNAPII=RNA polymerase II, TF=transcription factor).
Angew. Chem. Int. Ed. 50 (2011) 9788-9790
Direkte Beobachtung einzelner RNA-Polymerasen beim Ablesen eines endogenen Gens in einer lebenden Hefezelle
Neueste technologische Fortschritte in der Abbildung lebender Zellen haben es nun ermöglicht, die Entstehung einzelner mRNA-Moleküle bei der Transkription eines endogenen Hefegens zu beobachten. Eine quantitative Fluktuationsanalyse der fluoreszenzmarkierten mRNA-Moleküle gibt Einblicke in die Kinetik der Transkriptionsinitiation und die Dynamik von mRNA-Verlängerung und Termination (siehe Bild; GFP = Grün fluoreszierendes Protein, PP7 ist ein Bakteriophagen-Hüllprotein, RNAPII = RNA Polymerase II, TF = Transkriptionsfaktor).
Angew. Chem. 123 (2011) 9962-9964
J.R. Moffitt, C. Osseforth, and J. Michaelis
Time-gating improves the spatial resolution of STED microscopy
Stimulated-emission depletion (STED) microscopy improves image resolution by encoding additional spatial information in a second stimulated-decay channel with a spatially-varying strength. Here we demonstrate that spatial information is also encoded in the fluorophore lifetime and that this information can be used to improve the spatial resolution of STED microscopy. By solving a kinetic model for emission in the presence of a time-varying STED pulse, we derive the effective resolution as a function of fluorophore lifetime and pulse duration. We find that the best resolution for a given pulse power is achieved with a pulse of infinitesimally short duration; however, the maximum resolution can be restored for pulses of finite duration by time-gating the fluorescence signal. In parallel, we consider time-gating in the presence of a continuous-wave (CW) STED beam and find that time-gating produces theoretically unbounded resolution with finite laser power. In both cases, the cost of this improved resolution is a reduction in the brightness of the final image. We conclude by discussing situations in which time-gated STED microscopy (T-STED) may provide improved microscope performance beyond an increase in resolution.
Optics Express 19 (2011) 4242-4254
T. Lebold, J. Michaelis, and C. Bräuchle
The complexity of mesoporous silica nanomaterials unravelled by single molecule microscopy
Mesoporous silica nanomaterials are a novel class of materials that offer a highly complex porous network with nanometre-sized channels into which a wide amount of differently sized guests can be incorporated. This makes them an ideal host for various applications for example in catalysis, chromatography and nanomedicine. For these applications, analyzing the host properties and understanding the complicated host–guest interactions is of pivotal importance. In this perspective we review some of our recent work that demonstrates that single molecule microscopy techniques can be utilized to characterize the porous silica host with unprecedented detail. Furthermore, the single molecule studies reveal sample heterogeneities and are a highly efficient tool to gain direct mechanistic insights into the host–guest interactions. Single molecule microscopy thus contributes to a thorough understanding of these nanomaterials enabling the development of novel tailor-made materials and hence optimizing their applicability significantly.
Phys. Chem. Chem. Phys.,
Jiang, X., Musyanovych, A., Röcker, C., Landfester, K., Mailänder, V., Nienhaus, G. U.
Specific Effects of Surface Carboxyl Groups on Anionic Polystyrene Particles in their Interactions with Mesenchymal Stem Cells
Nanoscale 3 (2011) 2028-2035
Lunov, O., Zablotskii, V., Syrovets, T. Röcker, C., Tron, K., Nienhaus, G. U., & Simmet, T.
Modeling Receptor-mediated Endocytosis of Polymer-functionalized Iron Oxide Nanoparticles by Human Macrophages
Biomaterials 32 (2011) 547-55
Wacker, S. A., Alvarado, C., von Wichert, G., Knippschild, U., Wiedenmann, J., Clauss, K., Nienhaus, G. U., Hameister, H., Baumann, B., Borggrefe, T., Knöchel, W., Oswald, F.
RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J
EMBO J. 30 (2011) 43-56
J. Stigler, F. Ziegler, A. Gieseke, J.C.M. Gebhardt, and M. Rief
The Complex Folding Network of Single Calmodulin Molecules
Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.
Science 28 (2011) 512-516
C. Jung, P. Schwaderer, M. Dethlefsen, R. Köhn, J. Michaelis, and C. Bräuchle
Visualization of the self assembly of silica nanochannels reveals growth mechanism
Self assembled mesoporous structures with well ordered nanometer sized channels have enormous potential for applications in nanotechnology.
However, until now this potential could not be realised since the lack of understanding and of control of domain growth limited the obtainable domain sizes. Here, we present the real-time observation of domain growth by fluorescence polarization imaging and atomic force microscopy.
Nature Nanotechnology, DOI: 10.1038/NNANO0.2010.258
J. Michaelis, C. Bräuchle
Reporters in the nanoworld: diffusion of single molecules in mesoporous materials
Single molecules can be used as highly sensitive nanometer sized local reporters. Here, we review our recent work on the invesitgation of molecular diffusion in nanoscale networks and show how one can use the intensity of the emitted fluorescence, the polarisation of the fluorescence as well as the fluorescence emission spectra of single molecules to investigate details of the interaction with the molecule with its surroundings. Such findings have important implications also for novel drug delivery applications where a control of drug-carrier interactions can improve the controlled release of the drug.
Chemical Society Reviews, 39 (2010) 4731-4740
Lunov, O., Syrovets, T., Röcker, C., Tron, K., Nienhaus, G.U., Rasche, V., Mailänder, V., Landfester, K., & Simmet, T.
Lysosomal degradation of the carboxydextran shell of coated superparamagnetic iron oxide nanoparticles and the fate of professional phagocytes
Biomaterials, 31 (2010) 9015-9022
Jiang, X., Röcker, C., Hafner, M., Brandholt, S., Dörlich, R., & Nienhaus, G. U.
Endo- and Exocytosis of Zwitterionic Quantum Dot Nanoparticles by Living HeLa Cells
ACS Nano, 4 (2010) 6787-6797
Riedinger, A., Zhang, F., Dommershausen, F., Röcker, C., Brandholt, S., Nienhaus, G. U., Koert, U., Parak, W. J.
Ratiometric optical sensing of chloride ions with organic fluorophore - gold nanoparticle hybrids: a systematic study of design parameters and surface charge effects
Small 6 (2010) 2590-2597
Lunov, O., Syrovets, T., Büchele, B., Jiang, X., Röcker, C., Tron, K., Nienhaus, G. U., Walther, P., Mailänder, V., Landfester, K., & Simmet, T.
The effect of carboxydextran-coated superparamagnetic iron oxide nanoparticles on c-Jun N-terminal kinase-mediated apoptosis in human macrophages
Biomaterials, 31 (2010) 5063-5071
Lehmann, A. D., Parak, W. J., Zhang, F., Ali, Z., Roecker, C., Nienhaus, G. U., Gehr, P., & Rothen-Rutishauser, B.
Fluorescent-Magnetic Hybrid Nanoparticles Induce a Dose-Dependent Increase of the Pro-Inflammatory Response in Lung Cells in Vitro Correlated with Intracellular Localization
Small, 6 (2010) 753-762
Jiang, X., Dausend, J., Hafner, M., Musyanovych, A., Röcker, C., Landfester, K., Mailänder, V., & Nienhaus, G. U.
Specific Effects of Surface Amines on Polystyrene Nanoparticles in Their Interactions with Mesenchymal Stroma Cells
Biomacromolecules, 11 (2010) 748-753.
Jiang, X., Weise, S., Hafner, M., Röcker, C., Zhang, F., Parak, W. J., & Nienhaus, G. U.
Quantitative Analysis of the Protein Corona on FePt Nanoparticles formed by Transferrin Binding
J. R. Soc. Interface, 7 (2010) S5-S13.
M. Brunnbauer, F. Mueller-Planitz, S. Kösem, T.H. Ho, R. Dombi, J.C.M. Gebhardt, M. Rief, and Z. Ökten
Regulation of a Heterodimeric Kinesin-2 Through an Unprocessive Motor Domain that is Turned Processive by its Partner
Cilia are microtubule-based protrusions of the plasma membrane found on most eukaryotic cells. Their assembly is mediated through the conserved intraflagellar transport mechanism. One class of motor proteins involved in intraflagellar transport, kinesin-2, is unique among kinesin motors in that some of its members are composed of two distinct polypeptides. However, the biological reason for heterodimerization has remained elusive. Here we provide several interdependent reasons for the heterodimerization of the kinesin-2 motor KLP11/KLP20 of Caenorhabditis elegans cilia. One motor domain is unprocessive as a homodimer, but heterodimerization with a processive partner generates processivity. The “unprocessive” subunit is kept in this partnership as it mediates an asymmetric autoregulation of the motor activity. Finally, heterodimerization is necessary to bind KAP1, the in vivo link between motor and cargo.
Proc. Natl. Acad. Sci. 107 (2010) 10460-10465
J.C.M. Gebhardt, Z. Ökten, and M. Rief
The Lever Arm Effects a Mechanical Asymmetry of the Myosin-V – Actin Bond
Myosin-V is a two-headed molecular motor taking multiple ATP-dependent steps toward the plus end (forward) of actin filaments. At high mechanical loads, the motor processively steps toward the minus end (backward) even in the absence of ATP, whereas analogous forward steps cannot be induced. The detailed mechanism underlying this mechanical asymmetry is not known. We investigate the effect of force on individual single headed myosin-V constructs bound to actin in the absence of ATP. If pulled forward, the myosin-V head dissociates at forces twice as high than if pulled backward. Moreover, backward but not forward distances to the unbinding barrier are dependent on the lever arm length. This asymmetry of unbinding force distributions in a single headed myosin forms the basis of the two-headed asymmetry. Under load, the lever arm functions as a true lever in a mechanical sense.
Biophys J 98 (2010) 277-281
J.C.M. Gebhardt, T. Bornschlögl, and M. Rief
Full Distance-Resolved Folding Energy Landscape of one Single Protein Molecule
Kinetic bulk and single molecule folding experiments characterize barrier properties but the shape of folding landscapes between barrier top and native state is difficult to access. Here, we directly extract the full free energy landscape of a single molecule of the GCN4 leucine zipper using dual beam optical tweezers. To this end, we use deconvolution force spectroscopy to follow an individual molecule’s trajectory with high temporal and spatial resolution. We find a heterogeneous energy landscape of the GCN4 leucine zipper domain. The energy profile is divided into two stable C-terminal heptad repeats and two less stable repeats at the N-terminus. Energies and transition barrier positions were confirmed by single molecule kinetic analysis. We anticipate that deconvolution sampling is a powerful tool for the model-free investigation of protein energy landscapes.
Proc. Natl. Acad. Sci. USA 107 (2010) 2013-1018
J. Michaelis, A. Muschiolok, J. Andrecka, W. Kügel, and J. Moffitt
DNA based molecular motors
In our group we put a large emphasis on studying enzymes that move along DNA. The characteristics of these so called DNA motors are best investigated using single-molecule force spectroscopy such as AFM, magnetic tweezers or optical tweezers. In this review we discuss current experimental approaches and challenges. In order to understand the behaviour of DNA based molecular motors it is important to mechanically characterise DNA including force-extension behaviour and structural transitions. Finally we discuss experiments which shed light on the molecular mechansim of the bacteriophage Phi 29 DNA packaging motor as well as on RNA polymerases including our own previously unpublished data.
Physics of Life Reviews, 6 (2009) 250-266
J. Andrecka, B. Treutlein, M. Izquierdo Arcusa, A. Muschielok, R. Lewis, A. Cheung, P. Cramer, and J. Michaelis
Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex
Using our recently developed Nano-Positioning System we map the position of dye molecules attached to seven different locations along the nontemplate strand in Pol II elongation complex. The quality of the data allows us to build a model for the pathway of the nontemplate and upstream DNA thus completing our picture of the Pol II elongation complex.
The NPS analysis software used for this worl as well as a detailed description can be downloaded here:
NPS software and documentation
Nucleic Acids Research, 37 (2009) 5803-5809
T. Lebold, C. Jung, J. Michaelis, and C. Bräuchle
Nanostructured silica materials as drug-delivery systems for doxorubicin: single molecule and cellulare studies
We apply mesoporous thin silica films with nanometer-sized pores as drug carriers and incorporate the widely used anti-cancer drug Doxorubicin. Drug dynamics inside the nanopores is controlled by pore size and surface modification. This study demonstrates that mesoporous silica nanomaterials can provide solutions for current challenges in nanomedicine.
Nanoletters, 9 (2009) 2877-2883
F. Feil, C. Jung, J. Kirstein, J. Michaelis, C. Li, F. Nolde, K. Müllen, and C. Bräuchle
Diffusional and orientational dynamics of various single terylene diimide conjugates in mesoporous materials
Three structurally different TDI derivatives allowed studying the influence of the molecular structure of the guest on the translational diffusion behaviour in the hexagonal phase and the lamellar phase of mesoporous silica. In the lamellar phase, the differences between the three guests are quite dramatic. First, two populations of diffusing molecules – one with parallel orientation of the molecules to the lamellae and the other with perpendicular orientation – could be observed for two of the TDI derivatives. These populations differ drastically in their translational diffusion behaviour. Depending on the TDI derivative, the ratio between the two populations is different. Additionally, switching between the two populations was observed. These data provide new insights into host–guest interactions like the influence of the molecular structure of the guest molecules on their diffusional as well as on their orientational behaviour in structurally confined guest systems.
Microporous and Mesoporous Materials, 125 (2009) 70-78
T. Lebold, L.A. Mühlstein, J. Blechinger, M. Riederer, H. Amenitsch, R. Köhn, K. Peneva, K. Müllen, J. Michaelis, C. Bräuchle, and T. Bein
Tuning single-molecule dynamics in functionalized mesoporous silica
The diffusion dynamics of single molecules is very sensitive to variations in the local surrounding. In confined geometries such as the nanometer sized channels of mesoporous systems diffusion can be controlled by a careful design of the channel surface. We demonstrate this effect by using different organic modifications covalently linked to the walls of the nanometer sized channels which drastically effect the mobility of the TDI molecules difusing through the system.
Chemistry - A European Journal, 15 (2009) 1661-1672
C. Jung, N. Ruthardt, R. Lewis, J. Michaelis, B. Sodeik, F. Nolde, K. Peneva, K. Müllen, and C. Bräuchle
Photophysics of New Water-SolubleTerrylenediimides Derivates and Applications in Biology
Three new water-soluble TDI derivatives are investigated for membrane labelling, DNA labelling, and virus labelling. All three dye molecules are extremly photo-stable and are therefore ideally suited for applications where chromphores need to be investigated over extended periods of time.
ChemPhysChem, 10 (2009) 180-190
Röcker, C., Pötzl, M., Zhang, F., Parak, W. J., & Nienhaus, G. U.
A Quantitative Fluorescence Study of Protein Monolayer Formation on Colloidal Nanoparticles
Nature Nanotechnology, 4 (2009) 577-580.
Arhel, N., Lehmann, M., Clauss, K., Nienhaus, G. U., Piguet, V., & Kirchhoff, F.
The Inability to Disrupt the Immunological Synapse distinguishes HIV-1 from most other Primate Lentiviruses
J. Clin. Invest. 119 (2009) 2965-2975.
Kredel, S., Oswald, F., Nienhaus, K., Deuschle, K., Röcker, C., Wolff, M., Heilker, R., Nienhaus, G. U., & Wiedenmann, J.
mRuby, a Bright Monomeric Red Fluorescent Protein for Labeling of Subcellar Structures
PLoS ONE 4 (2009) e4391.
Bismuto, E., Di Maggio, E., Pleus, S., Sikor, M., Röcker, C., Nienhaus, G. U., & Lamb, D. C.
Molecular Dynamics Simulation of Acidic Compact State of Apomyoglobin from Yellowfin Tuna
Proteins: Structure, Function and Bioinformatics 74 (2009) 273-290.
J.C.M. Gebhardt and M. Rief
Force Signaling in Biology
Many processes in our body, like muscle contraction, cell locomotion and division, or transport processes, need force-producing actuators such as molecular motors. In turn, biological systems can also sense mechanical forces. Examples are the sense of touch, hearing, and the strengthening of muscle tissues upon physical exercise. In these cases, force triggers a biochemical signal cascade, but the mechanisms by which forces affect biomolecular conformation and biochemical signaling have long remained elusive. The development of ultrasensitive instruments for nanomanipulation - such as atomic force microscopy and optical and magnetic tweezer - has allowed the effect of forces on protein conformation and function to be probed at the single-molecule level (1–4).
Science 324 (2009) 1278-1280
T. Bornschlogl, J.C.M. Gebhardt, and M. Rief
Designing the Folding Mechanics of Coiled Coils
Naturally occurring coiled coils are often not homogeneous throughout their entire structure but rather interrupted by sequence discontinuities and non-coiled-coil-forming subsegments. We apply atomic force microscopy to locally probe the mechanical folding/unfolding process of a well-understood model coiled coil when unstructured subsegments with different sizes are added. We find that the refolding force decreases from 7.8 pN with increasing size of the added unstructured subsegment, while the unfolding properties of the model coiled coil remain unchanged. We show that this behavior results from the increased size of the nucleation seed which has to form before further coiled-coil folding can proceed. Since the nucleation seed size is linked to the width of the energetic folding barrier, we are able to directly measure the dependence of folding forces on the barrier width. Our results allow the design of coiled coils with designated refolding forces by simply adjusting the nucleation seed size.
Chemphyschem 10 (2009) 2800-2804
A. Muschielok, J. Andrecka, A. Jawhari, F. Brückner, P. Cramer, and J. Michaelis
A nano-positioning system for macromolecular structural analysis
By combining single pair FRET data, x-ray crystallographical data and statistical data analysis we have developed a novel method for determining the position of a flexible domain inside of a macromolecule/macromolecular complex. The method shares some similarities to GPS, the global positioning system, which is why we called it NPS, Nano Positioning System.
We developed a easy to use NPS software which can be downloaded for free:
NPS software and documentation
Nature Methods, 5 (2008 ) 965-971
R. Lewis,H. Dürr, K.-P. Hopfner, and J. Michaelis
Conformational changes of a Swi2/Snf2 ATPase during its mechano-chemical cycle
We investigate the conformation of an archaebacterial homolgue of Rad54 in a nucleotide and DNA dependent fashion. Our results allow us to propose a minimal mechano-chemical scheme for the DNA translocation function of this enzyme. It will be interesting to see how well this scheme can describe the function of other members of this large superfamily of DNA translocases.
Nucleic Acids Research, 36 (2008) 1881-1890
C. Jung, J. Kirstein, B. Platschek, T. Bein, M. Budde, I. Frank, K. Mullen, J. Michaelis, and C. Bräuchle
Diffusion of oriented single molecules with switchable mobility in networks of long unidimensional nanochannels
Single dye molecules are incorporated as guest within the pores of mesoporous systems. We study the diffusion of the molecules within the nanometer sized channels. By accurately fitting the point spread function of the dye molecules we can map the trajectories of the molecules and directly observe jumps from one channel to the next.
J. of the American Chemical Society, 130 (2008) 1638-1648
J. Andrecka, R. Lewis, F. Brückner, E. Lehmann, P. Cramer, and J. Michaelis
Single-molecule tracking of mRNA exiting from RNA polymerase II
Single-molecule FRET is used to map the position of the nascent RNA in RNA polymerase II elongation complexes. In the paper we use a novel hybrid approach of single-molecule fluorescence data and high resolution structural models. In analogy to satellites in the global positioning system (GPS), single-dye molecules act as nanoscopic reporters at known locations. Single-pair FRET can than be used to accurately determine a desired position of a flexible domain in large protein-nucleic acid complexes.
PNAS, 105 (2008) 135-140
P. Schwaderer, E. Funk, F. Achenbach, J. Weiss, C. Bräuchle, and J. Michaelis
Single-molecule measurement of the strength of a siloxane bond
We investigate the mechanical stability of siloxane polymers by rupturing a single molecule using an AFM cantilever. Histograms of many rupture events can be analysed with dynamic force spectroscopy, allowing us to reveal details of the underlying potential energy landscape.
Langmuir, 24 (2008) 1343-1349
Tron, K., Manolov, D. E., Röcker, C., Kächele, M., Torzewski, J., & Nienhaus, G. U.
C-reactive Protein Specifically Binds to FcγRI on a Macrophage-like Cell Line
Eur. J. Immunol., 38 (2008) 1414-1422.
S. Grimm, D. Tabatabai, A. Scherer, J. Michaelis, and I. Frank
Chromophore localization in conjugated polymers: molecular dynamics simulation
Using molecular dynamics simulations we show that the size of chromophores in long conjugated polymers is limited by conformational changes at elevated temperatures in contrast to quantum chemical calculations at zero temperature which predict a chromophore extending over the whole polymer chain.
J. of Phys. Chem., B 111 (2007) 12053-12058
C. Jung, C. Hellriegel, B. Platschek, D. Wöhrle,T. Bein, J. Michaelis, and C. Bräuchle
Simultaneous measurement of orientational and spectral dynamics of single molecules in nanostructured host-guest materials
We compare the behaviour of singel dye molecules inside of two different nanostructured materials. While in one system the molecule fits tightly into the pores, and therefore rotational diffusion is hindered, a larger pore diameter allows for the investigation of rotational jumps. In the latter system the molecule acts as an active reporter experiencing local inhomogeneities of the surrounding medium.
J. of the American Chem. Soc., 129 (2007) 5570-5579
C. Jung, C. Hellriegel, J. Michaelis, and C. Bräuchle
Single-molecule traffic in nanoporous materials
Single-molecule traffic in mesoporous materials: translational, orientational and spectral dynamics.
We investigate the translational, orientational and spectral dynamics of single dye molecules inside of the channels of a nanoporous network.
Advanced Materials, 19 (2007) 956-960
T. Hugel, J. Michaelis, C. Hetherington, et al.
Experimental test of connector rotation during DNA packaging into bacteriophage φ29 capsids
The bacteriophage φ29 uses a molecular motor to drive its genome into a preformed protein capsid. The central part of this molecular motor is formed by a ring of twelve proteins called the connector. Symmetry and structural arguments have let to the so called rotation hypothesis, i.e. a rotation of the connector is used to drive the DNA into the capsid, which for the last 30 years has been the most prominent model for the function of this motor. We have tested this hypothesis using single-molecule fluorescence polarisation and could show, that the connector does not rotate during packaging. Therefore a new model for motor function had to be developed.
PLOS Biology, 5 (2007) 558-567
The paper was highlighted by the editorial team of PLOS Biology in this commentary:
Does Bacteriophage φ29 Package its DNA with a twist?
K.-P. Hopfner and J. Michaelis
Mechanism of nucleic acid translocases: lessons from structural biology and single-molecule biophysics
Enzymes that translocate nucleic acids using ATP hydrolysis form a auperfamily with quite distinct biological functions ranging from DNA and RNA helicase, viral genome packaging motors, enzymes involved in DNA repair, chromatin remodeling ATPases and others. In this review we discuss recent structural and single-molecule results and how they influence our current understanding of translocases.
Current Opinion in Structural Biology, 17 (2007) 87-95
Glaschick, S., Röcker, C., Deuschle, K., Wiedenmann, J., Oswald, F., Mailänder, V., & Nienhaus, G. U.
Axial Resolution Enhancement by 4Pi Confocal Fluorescence Microscopy with Two-Photon Excitation
J. Biol. Phys., 33 (2007) 433-443.
Ivanchenko, S., Glaschick, S., Röcker, C., Oswald, F., Wiedenmann, J., & Nienhaus, G. U.
Two-photon Excitation and Photoconversion of EosFP in Dual-color 4Pi Confocal Microscopy
Biophys. J. 92 (2007) 4451-4457.
Röcker, C., Manolov, D. E., Kuzmenkina, E. V., Tron, K., Slatosch, H., Torzewski, J., & Nienhaus, G. U.
Affinity of C-Reactive Protein towards FcγRI is Strongly Enhanced by the γ-Chain
Am J. Pathol. 170 (2007) 755-763.
G. Cappello, P. Pierobon, C. Symonds, L. Busoni, J.C.M. Gebhardt, M. Rief, and J. Prost
Myosin-V Stepping Mechanism
We observe the myosin V stepping mechanism by traveling wave tracking. This technique, associated with optical tweezers, allows one to follow a scattering particle in a two-dimensional plane, with nanometer accuracy and a temporal resolution in the microsecond range. We have observed that, at the millisecond time scale, the myosin V combines longitudinal and vertical motions during the step. Because at this time scale the steps appear heterogeneous, we deduce their general features by aligning and averaging a large number of them. Our data show that the 36-nm step occurs in three main stages. First, the myosin center of mass moves forward 5 nm; the duration of this short prestep depends on the ATP concentration. Second, the motor performs a fast motion over 23 nm; this motion is associated to a vertical movement of the myosin center of mass, whose distance from the actin filament increases by 6 nm. Third, the myosin head freely diffuses toward the next binding site and the vertical position is recovered. We propose a simple model to describe the step mechanism of the dimeric myosin V.
Proc. Nat. Acad. Sci. 104 (2007) 15328-15333
2006 and earlier
J. Michaelis and N. Ruthardt
Fluoereszierende Proteine zeigen den Weg durch die Zelle
Introductory review of single-molecule fluorescence, fluorescent proteins and live cell imaging (in German).
Nachrichten aus der Chemie, 54 (2006) 1222-1225
Nienhaus, G. U., Nienhaus, K., Hölzle, A., Ivanchenko, S., Renzi, F., Oswald, F., Wolff, M., Schmitt, F., Röcker, C., Vallone, B., Weidemann, W., Heilker, R., Nar, H., & Wiedenmann, J.
Photoconvertible Fluorescent Protein EosFP: Biophysical Properties and Cell Biology Applications
Photochem. Photobiol., 82 (2006) 351-358.
Owen, R. J., Heyes, C. D., Knebel, D., Röcker, C., & Nienhaus, G. U.
An Integrated Instrumental Setup for the Combination of Atomic Force Microscopy with Optical Spectroscopy
Biopolymers, 82 (2006) 410-414.
J.C.M. Gebhardt, A.E.-M. Clemen, J. Jaud, and M. Rief
Myosin-V is a Mechanical Ratchet
Myosin-V is a linear molecular motor that hydrolyzes ATP to move processively toward the plus end of actin filaments. Motion of this motor under low forces has been studied recently in various single-molecule assays. In this paper we show that myosin-V reacts to high forces as a mechanical ratchet. High backward loads can induce rapid and processive backward steps along the actin filament. This motion is completely independent of ATP binding and hydrolysis. In contrast, forward forces cannot induce ATP-independent forward steps. We can explain this pronounced mechanical asymmetry by a model in which the strength of actin binding of a motor head is modulated by the lever arm conformation. Knowledge of the complete force–velocity dependence of molecular motors is important to understand their function in the cellular environment.
Proc. Nat. Acad. Sci. 103 (2006) 8680-8685
A. Scherer, C. Zhou, J. Michaelis, C. Bräuchle, and A. Zumbusch
Intermolecular interactions of polymer molecules determined by single-molecule force spectroscopy
Polymer molecules tend to aggregate in bad solvent conditions. In this work we demonstrate that by pulling such a collapsed polymer bundle into solution, we can examine the inter-molecular interactions and the solvation energy of a single-molecule. This technique therefore provides the possibility for studying interaction and solvent quality on the single-molecule level.
Macromolecules, 38 (2005) 9821-9825
Y. R. Chemla, K. Aathavan, J. Michaelis, S. Grimes, P. J. Jardine, D. L. Anderson, and C. Bustamante
Mechanism of force generation of a viral DNA packaging motor
Molecular motors couple chemical energy to produce mechanical work and uni-directional motion. Here we study how the chemical energy is coupled to mechanical work in the case of the bacteriophage phi29 portal packaging motor. The paper describes single-molecule force spectroscopy methods using optical tweezers, as well as bulk bio-chemical assays. The results allowed us to formulate a model for packaging that is illustrated in a small animation.
Download the animation (850kB)
Cell, 122 (2005) 683-692
A. Hards, C. Zhou, M. Seitz, C. Bräuchle, and A. Zumbusch
Simultaneous AFM manipulation and fluorescence imaging of single DNA strands
Combining single-molecule manipulation and high sensitivity fluorescence measurements is one of the recent acchievements of the lab. This paper discuss the manipulation of dye labelled DNA molecules. The molecules are stretched with the AFM and at high forces chain ruptures are observed. This work was supervised by Andreas Zumbusch.
ChemPhysChem, 6 (2005) 534-540
Anikin, K., Röcker, C., Wittemann, A., Wiedenmann, J., Ballauff, M., & Nienhaus, G. U.
Polyelectrolyte-Mediated Protein Adsorption: Fluorescent Protein Binding to Individual Polyelectrolyte Nanospheres
J. Phys. Chem., B 109 (2005) 5418-5420.
Ivanchenko, S., Röcker, C., Oswald, F., Wiedenmann, J., & Nienhaus, G. U.
Targeted Green-to-Red Photoconversion of EosFP, a Fluorescent Marker Protein
J. Biol. Phys., 31 (2005) 249-259.
Rieger, R., Röcker, C., & Nienhaus, G. U.
Fluctuation Correlation Spectroscopy for the Advanced Physics Laboratory
Am. J. Phys., 73 (2005) 1129-1134.
Wiedenmann, J., Vallone, B., Renzi, F., Nienhaus, K., Ivanchenko, S., Röcker, C., & Nienhaus, G. U.
The Red Fluorescent Protein eqFP611 and its Genetically Engineered Dimeric Variants
J. Biomed. Opt., 10 (2005) 014003 (7 pages).
Amirgoulova, E. V., Groll, J., Heyes, C. D., Ameringer, T., Röcker, C., Möller, M., & Nienhaus, G. U.
Biofunctionalized Polymer Surfaces Exhibit Minimal Interaction towards Immobilized Proteins
ChemPhysChem, 5 (2004) 552-555.
Groll, J., Amirgoulova, E. V., Ameringer, T., Heyes, C. D., Röcker, C., Möller, M., & Nienhaus, G. U.
Biofunctionalized, Ultrathin Coatings of Cross-Linked Star-Shaped Poly(ethylene oxide) Allow Reversible Folding of Immobilized Proteins
J. Am. Chem. Soc., 126 (2004) 4234-4239.
Manolov, D. E., Röcker, C., Hombach, V., Nienhaus, G. U., & Torzewski, J.
Ultrasensitive Confocal Fluorescence Microscopy of C-Reactive Protein Interacting with FcγRIIa
Arteriosclerosis, Thrombosis and Vascular Biology, 24 (2004) 2372-2377.
Schenk, A., Ivanchenko, S., Röcker C., Wiedenmann, J., & Nienhaus, G. U.
Photodynamics of Red Fluorescent Proteins Studied by Fluorescence Correlation Spectroscopy
Biophys. J., 86 (2004) 384-394.
Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Röcker, C., Salih, A., Spindler, K.-D., & Nienhaus, G. U.
EosFP, A Fluorescent Marker Protein with UV-Inducible Green-to-Red Fluorescence Conversion
Proc. Natl. Acad. Sci. USA, 101 (2004) 15905-15910.
Wiedenmann, J., Vallone, B., Renzi, F., Nienhaus, K., Ivanchenko, S., Röcker, C., & Nienhaus, G. U.
Dimeric Variants of the Red Fluorescent Protein eqFP611 Generated by Site-Directed Mutagenesis
Proc. SPIE, 5329 (2004) 23-29.
P. Persson, J.C.M. Gebhardt, and S. Lunell
The Smallest Possible Nanocrystals of Semiionic Oxides
General bonding principles are used to predict the structure of individual nanocrystals in nanocrystalline materials with semiionic bonding. The relationship between the general principles and actual nanocrystal structures is demonstrated using titanium dioxide in the anatase form. The proposed nanocrystals simultaneously fulfill strict criteria of stoichiometry, high coordination, and balanced charge distribution. The smallest such nanocrystals are remarkably simple, e.g., consisting of less than 100 atoms in anatase. According to computer simulations, these nanocrystals show strong quantum size effects, while other clusters of similar size instead show typical defect characteristics.
J. Phys. Chem. B 107 (2003) 3336-3339
Wiedenmann, J., Schenk, A., Röcker, C., Girod, A., Spindler, K.-D., & Nienhaus, G. U.
A Far-Red Fluorescent Protein with Fast Maturation and Low Oligomerization Tendency from Entacmaea quadricolor (Anthozoa, Actinaria)
Proc. Natl. Acad. Sci. USA, 99 (2002) 11646-11651
V. Sandoghdar, J. Michaelis, C. Hettich, C. Schmidt, J. Zitzmann, and S. Kühn
Results and thoughts on optical microscopy using a single-molecule probe
This article discusses recent acchievements in near-field optical microscopy.
Molecules, 2 (2001) 277-281
Chan, V. Z. H., Codd, S. L., van der Helm, M. J., Spatz, J. P., Röcker, C., Nienhaus, G. U., Levi, S., van Veggel, F. C. J. M., Reinhoudt, D. N., & Möller, M.
Sub-10 nm Gold Nanoarrays for Tethering Single Molecules
in: Synthesis, Functional Properties and Applications of Nanostructures
eds.: H. W. Hahn, D. L. Feldheim, C. P. Kubiak, R. Tannenbaum, R. W. Siegel, MRS Publications
Materials Research Society Symposium Proceedings, Vol. 676, Pittsburgh PA (2001) Y4.4.1-Y4.4.6
J. Michaelis, C. Hettich, J. Mlynek, V. Sandoghdar
Optical microscopy using a single-molecule light source
This paper describes a new approach to overcome the resolution limits of optical microscopy. Here we are using the ultimate lightsource a single-molecule for the illumination of a test-pattern. In the optical near-field the resolution is limited by the size of the light source and therefore the smaller the light source, the higher the resolution. Here we are using the ultimate lightsource a single-molecule for the illumination of a test-pattern.
Nature, 405 (2000) 325-328
Lamb, D.C., Schenk, A., Röcker, C. & Nienhaus, G.U.
Determining chemical rate coefficients using time-gated fluorescence correlation spectroscopy
J. Phys. Org. Chem., 13 654-658 (2000).
Lamb, D. C., Schenk, A., Röcker, C., Scalfi-Happ, C., & Nienhaus, G. U.
Sensitivity Enhancement in Fluorescence Correlation Spectroscopy of Multiple Species Using Time-Gated Detection
Biophys. J., 79 (2000), 1129-1138
J. Michaelis, C. Hettich, A. Zavats, B. Eiermann, J. Mlynek, and V. Sandoghdar
A single molecule as a probe of optical intensity distribution
A single molecule can be used as a poin-like reporter for the surrounding lightfield. Here we use a single dye molecule to map the intensity distribution of standing wave. This technique might also be used for high resolution optical microscopy, due to the sharp gradients in the cos^2 functions of the standing wave.
Optics Letters, 24 (1999) 581-583
Wiedenmann, J., Röcker, C. & Funke, W.
The morphs of Anemonia aff. Sulcata (Cnidaria, Anthozoa) in particular consideration of the ectodermal pigments Verhandlungen der Gesellschaft für Ökologie 29, 497-503 (1999).
Röcker, C., Heilemann, A. & Fromherz, P.
Time-resolved fluorescence of a hemicyanine dye: dynamics of rotamerism and resolvation
J. Phys. Chem., 100, 12172-12177 (1996).
Fromherz, P. & Röcker, C.
Staining of biomembranes with amphiphilic hemicyanine dyes
Ber. Bunsenges. Phys. Chem,. 98, 128-131 (1994).
Fromherz, P., Röcker, C. & Rüppel, D.
From discoid micelles to spherical vesicles: the concept of edge activity
Faraday Discuss. Chem. Soc., 81, 39-48 (1986).