Publications
2012
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 10.1093/nar/gks267
The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ singlemolecule Förster 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.
Barbara Treutlein, Adam Muschielok, Joanna Andrecka, Anass Jawhari, Claudia Buchen, Dirk Kostrewa,
Friederike Hög, Patrick Cramer, and Jens Michaelis
Molecular Cell
(2012), doi:10.1016/j.molcel.2012.02.008
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.
Wolfgang Kügel, Adam Muschielok and Jens Michaelis
ChemPhysChem. 2012
2011
Florian Feil, Sergej Naumov, Jens Michaelis, Rustem Valiullin, Dirk Enke, Jörg Kärger,
and Christoph Bräuchle
Angew. Chem. 2011, 123; DOI: 10.1002/anie.201105388
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.
Adam Muschielok, Jens Michaelis
Journal physical chemistry B 2011, 115, 11927-11937
Supplementary Material
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).
Barbara Treutlein, Jens Michaelis
Angew. Chem. Int. Ed. 2011, 50, 9788 – 9790; DOI: 10.1002/anie.201103809
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.
Dina Grohmann, Julia Nagy, Anirban Chakraborty, Daniel Klose, Daniel Fielden, Richard H. Ebright,
Jens Michaelis, and Finn Werner
Molecular Cell 43, 263-274, July 22, 2011
Supplemental Information
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.
Jeffrey R. Moffitt, Christian Osseforth, and Jens Michaelis
Optics Express (2011) Vol. 19, No. 5
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
2010
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.
Christophe Jung, Peter Schwaderer, Mark Dethlefsen, Ralf Köhn, Jens Michaelis* and Christoph Bräuchle*,
Nature Nanotechnology accepted *(cooresponding authors).
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.
Michaelis, C. Bräuchle, Chemical Society Reviews, 39, 4731-4740 (2010)
2009
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.
J.
Michaelis, A. Muschielok, J. Andrecka, W. Kügel, and J.
Moffitt, Physics of Life Reviews 6, 250-266 (2009)
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
J.
Andrecka, B. Treutlein, M.
Izquierdo Arcusa, A. Muschielok, R. Lewis, A. Cheung, P. Cramer
and J. Michaelis, Nucleic Acids Research, 37, 5803-5809 (2009).
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 nano-medicine.
T. Lebold, C. Jung, J.
Michaelis and C.
Bräuchle, Nanoletters, 9, 2877-2883 (2009).
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.
F. Feil, C. Jung, J.
Kirstein, J. Michaelis, C. Li, F. Nolde, K. Müllen and C.
Bräuchle,
Microporous and Mesoporous Materials, 125, 70-78 (2009).
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.
T. Lebold, L.A.
Mühlstein, J. Blechinger, M. Riederer, H. Amenitsch, R.
Köhn, K. Peneva, K. Müllen, J. Michaelis, C.
Bräuchle and T. Bein, Chemistry - A European Journal 15,
1661-1672 (2009).
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.
C. Jung, N. Ruthardt, R.
Lewis, J. Michaelis, B. Sodeik, F. Nolde, K. Peneva, K. Müllen
and C. Bräuchle, ChemPhysChem
10, 180-190 (2009).
2008
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
A.
Muschielok, J. Andrecka, F. Brückner, A. Jawhari, P. Cramer
and J. Michaelis, Nature Methods 5, 965-971 (2008).
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.
R. Lewis,
H. Duerr, K.-P. Hopfner and J. Michaelis, Nucleic Acids Research 36,
1881-1890 (2008).
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.
C. Jung, J.
Kirstein, B. Platschek, T. Bein, M. Budde, I. Frank, K. Mullen, J.
Michaelis and C. Braeuchle, Journal of the American Chemical Society
130, 1638-1648 (2008).
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.
J.
Andrecka, R. Lewis, F. Brueckner, E. Lehmann, P. Cramer and J.
Michaelis, PNAS 105, 135-140 (2008).
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.
P.
Schwaderer, E. Funk, F. Achenbach, J. Weiss, C. Braeuchle and J.
Michaelis, Langmuir 24, 1343-1349 (2008).
2007
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.
S. Grimm,
D. Tabatabai, A. Scherer, J. Michaelis and I. Frank, Journal of
Physical Chemistry B 111, 12053-12058 (2007).
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.
C. Jung, C. Hellriegel, B.
Platschek, D. Woehrle, T. Bein, J. Michaelis and C. Braeuchle,
Simultaneous measurement of orientational and spectral dynamics
of single molecules in nanostructured host-guest materials, Journal of
the American Chemical Society 129, 5570-5579 (2007).
We investigate the translational, orientational and spectral dynamics
of single dye molecules inside of the channels of a nanoporous network.
C. Jung, C. Hellriegel, J.
Michaelis and C. Braeuchle, Single-Molecule Traffic in Mesoporous
Materials: Translational, Orientational and Spectral Dynamics, Advanced
Materials 19, 956-960 (2007).
The bacteriophage Phi29 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.
T. Hugel,
J. Michaelis, C. Hetherington et al., Experimental Test of Connector
Rotation during DNA Packaging into Bacteriophage Phi29 Capsids, PLOS
Biology 5, 558-567 (2007).
The paper was highlighted by the editorial team of PLOS Biology in this
Commentary:
Does
Bacteriophage Phi29 Package its DNA with a twist?
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.
K.-P. Hopfner and J.
Michaelis, Current Opinion in Structural Biology, 17,
87-95 (2007).
2006 and earlier
Introductory review of single-molecule fluorescence, fluorescent
proteins and live cell imaging (in German).
J. Michaelis and N. Ruthardt,
Nachrichten aus der Chemie 54, 1222(2006)
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.
A. Scherer, C. Zhou, J. Michaelis, C. Braeuchle and A. Zumbusch,
Macromolecules 38, 9821(2005)
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)
Y. R.
Chemla, K. Aathavan, J. Michaelis, S. Grimes., P. J. Jardine, D. L.
Anderson and C. Bustamante, Cell 122, 683(2005)
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.
J. Michaelis, C. Hettich, J.
Mlynek, V.Sandoghdar, Nature 405, 325 (2000)
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.
J. Michaelis, C. Hettich, A.
Zayats, B. Eiermann, J. Mlynek and V. Sandoghdar, Optics Letters 24,
581 (1999)
This article discusses recent acchievements in near-field optical
microscopy.
V. Sandoghdar, J. Michaelis,
C. Hettich, C. Schmidt, J. Zitzmann and S. Kuehn, Single Molecules 2,
277 (2001)
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
.
A. Hards, C. Zhou, M. Seitz,
C. Bräuchle and A. Zumbusch, ChemPhysChem 6,
534 (2005)