Single-molecule microscopy study of nano-systems

In this work, techniques were developed and used to study the
properties of molecules on a single-molecule level. Single-molecule
techniques have the major advantage, that in contrast to ensemble
measurements, they allow a detailed insight on the distribution and
dynamics of single molecules without averaging over subpopulations.
The use of Total Internal Reflection Fluorescence Microscopy
(TIRFM) in combination with single-pair Förster Resonance Energy
Transfer (spFRET) and Alternating Laser Excitation (ALEX) allows
the identification of molecular-states by making quantitative
measurements of distances in the Ångström range. The development of
highly sensitive photon detectors and the use of versatile labeling
techniques with photostable (synthetic or genetically-encoded)
fluorophores, extended the application of TIRF microscopy to in
vitro and live-cell experiments. Despite reducing the complexity of
biological systems down to the single-molecule level, functions of
individual molecules and interactions between them can be very
sophisticated and challenging to analyze. Using information theory
based methods, e.g. HMM, the dynamics extracted from
single-molecule data was used to illuminate protein interactions
and functions. The highly regulated process of gene transcription
plays a central role in living organisms. The TATA-box Binding
Protein (TBP) is a Transcription Factor (TF) that mediates the
formation of the Pre-Initiation Complex (PIC). The lifetime of TBP
at the promoter site is controlled by the Modulator of
transcription 1 (Mot1), an essential TBP-associated ATPase involved
in repression and in activation of transcription. Based on ensemble
measurements, various models for the mechanism of Mot1 have been
proposed. However, little is known about how Mot1 liberates TBP
from DNA. Using TIRF microscopy, the conformation and interaction
of Mot1 with the TBP/DNA complex were monitored by spFRET. In
contrast to the current understanding of how Mot1 works, Mot1 bound
to the TBP/DNA complex is not able to directly disrupt the TBP/DNA
complexes by ATP hydrolysis. Instead, Mot1’s ATPase activity
induces a conformational change in the complex. The nature of this
changed, "primed", conformation is the change of the bending
dynamics of the DNA. The results presented in this work suggest a
model in which this primed conformation is a destabilized TBP/DNA
complex. The interaction with an additional Mot1 molecule is
required in order to liberate TBP from DNA. The effect of Mot1 on
the DNA dynamics is TBP binding orientation specific. Mot1 effects
on the DNA bending dynamics are strongest for molecules where TBP
is bound in the inverted binding orientation. The specificity of
Mot1’s regulation of DNA bending dynamics suggests that Mot1
preferably "primes" TBP bound in the inverted binding orientation.
The mechanistic insight into the interaction of Mot1 with the
TBP/DNA complex serves as a framework for understanding the role of
Mot1 in gene up- and down-regulation. In a second project, the same
single-molecule techniques were used to fabricate and evaluate
self-assembled optically controllable, nanodevices. Based on the
specificity of Watson-Crick base pairing, DNA was used as a
scaffold to position different fluorophores with nanometer
accuracy. The functionality of these nanodevices was expanded by
making them optically addressable by incorporation of the
switchable fluorescent protein Dronpa. Two functions have been
demonstrated: Signal enhancement using Optical Lock-In Detection
(OLID) and pH sensing in a live-cell environment.