Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones

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.