Folding of newly synthesized proteins is an essential part of protein biosynthesis and misfolding

can result in protein aggregation which can also lead to several severe diseases. Protein folding is a

highly heterogeneous process and rarely populated intermediate states may play an important role.

Single-molecule techniques are ideally suited to resolve these heterogeneities. In this thesis, I have

employed a variety of single-molecule fluorescence spectroscopy techniques to study protein folding

using model systems on different levels of complexity. The acidic compact state (A state) of Myo-

globin is used as a model system of a protein folding intermediate and is studied by a combination

of molecular dynamics (MD) simulations and several fluorescence spectroscopic techniques. Using

two-focus fluorescence correlation spectroscopy (FCS), it is shown that the A state is less compact

than the native state of myoglobin, but not as expanded as the fully unfolded state. The analysis of

exposed hydrophobic regions in the acidic structures generated by the MD simulations reveals poten-

tial candidates involved in the aggregation processes of myoglobin in the acidic compact state. These

results contribute to the understanding of disease-related fibril formation which may lead ultimately

to better treatments for these diseases.

A huge machinery of specialized proteins, the molecular chaperones, has evolved to assist protein

folding in the cell. Using single molecule fluorescence spectroscopy, I have studied several members

of this machinery. Single-pair fluorescence resonance energy transfer (spFRET) experiments probed

the conformation of the mitochondrial heat shock protein 70 (Hsp70), Ssc1, in different stages along

its functional cycle. Ssc1 has a very defined conformation in the ATP state with closely docked

domains but shows significantly more heterogeneity in the presence of ADP. This heterogeneity is due

to binding and release of ADP. The nucleotide-free state has less inter-domain contacts than the ATP

or ADP-bound states. However, the addition of a substrate protein decreases the interaction between

the domains even further simultaneously closing the substrate binding lid, showing that substrate

binding plays an active role in the remodeling of Ssc1. This behavior is strikingly different than in

DnaK, the major bacterial Hsp70. In DnaK, complete domain undocking in the presence of ADP

was observed, followed by a slight re-compaction upon substrate binding. These differences may

reflect tuning of Ssc1 to meet specific functions, i.e. protein import into mitochondria, in addition

to protein folding. Ssc1 requires the assistance of several cofactors depending on the specific task at

hand. The results of spFRET experiments suggest that the cofactors modulate the conformation of

Ssc1 to enable it to perform tasks as different as protein import and protein folding.

Downstream of Hsp70 in the chaperone network, the GroEL/ES complex is a highly specialized

molecular machine that is essential for folding of a large subset of proteins. The criteria that distin-

guish proteins requiring the assistance of GroEL are not completely understood yet. It is shown here

that GroEL plays an active role in the folding of double-mutant maltose binding protein (DM-MBP).

DM-MBP assumes a kinetically trapped intermediate state when folding spontaneously, and GroEL

rescues DM-MBP by the introduction of entropic constraints. These findings suggest that proteins

with a tendency to populate kinetically trapped intermediates require GroEL assistance for folding.

The capacity of GroEL to rescue proteins from such folding traps may explain the unique role of

GroEL within the cellular chaperone machinery.

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.

More than 90% of the worldwide population is infected with Epstein Barr virus (EBV). In general, the infection remains asymptomatic and the virus persists for life in latently infected B cells. During this state, viral activity is reduced to the maintenance of the viral genome such that EBV-infected cells are not efficiently recognized by the immune system. However, the first days of primary infection are characterized by a period of lytic gene expression that contributes to successful virus establishment (Kalla et al., 2010). The abundant expression of “foreign” protein renders the infected cell prone to immune recognition and requires viral counter-measures to evade immune responses.

The first part of this work focuses on the importance of two immune evasins of EBV during this early phase of infection: BCRF1 and BNLF2a, encoding the viral homologue of the anti-inflammatory cytokine IL-10 and a unique inhibitor of epitope loading on MHC I molecules, respectively. To study their impact on immune responses to freshly infected B cells, I generated recombinant EBV mutants, which are deficient in expressing either BCRF1 or BNLF2a or both. I observed significant immunological consequences during the first days of infection, but, unexpectedly, the MHC I surface pattern of infected B cells was not affected. Deficiency in BNLF2a expression in infected cells resulted in a drastically increased response of EBV-specific CD8+ T cells. BCRF1 did not participate in this effect, but instead severely impaired the cytokine response to viral infection and protected infected cells from elimination by NK/NKT cells. Thus, both immune evasins have important functions during the early phase of infection and are instrumental for the establishment of latency. Their balanced activity illustrates the perfect adaptation of the virus to the host’s immune system.

The second part of this work builds upon my initial observation that viral transcripts are present in the infected cell almost instantly following virus entry. I found that EBV particles represent the source of these transcripts as I identified them to contain viral RNA which is transferred to and expressed in the infected cell. I could further demonstrate that these packaged transcripts exert important biological functions in the infected cells by triggering the initial viral transcription program or acting as immune modulators. In sum, delivered RNAs apparently create a supportive environment that promotes virus establishment in infected B cells. Thus, despite EBV’s classification as a DNA virus, also packaged RNAs are apparently essential for its infectious nature.