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Biological
Questions and Systems
Mechanisms of Protein folding, Misfolding and
Aggregation
Assembly of complex biological machines
Pathways and mechanisms of siRNA-induced RNAi
Single-molecule
methods.
One important focus of the
laboratory is on developing fluorescence methods to measure intra- and
inter-molecular interactions and distances for molecules and complexes freely diffusing
in solution. The key motivation for developing these methods is to study
structural features of proteins, nucleic acids, and other biological systems,
while minimizing perturbations arising from surface-immobilization. In
addition, we also use imaging in a total-internal-reflection mode to monitor
longer molecular dynamics. Current
methods primarily use Förster energy transfer (FRET) and allow us to (i)
directly monitor properties of subpopulations of molecules in mixtures as a
function of time and under different solution conditions and (ii) monitor
transitions between different states of a system during equilibrium
fluctuations.(1,2)
Our single-molecule instrumentation
used for measurements on diffusing molecules consists of cw/pulsed lasers for
excitation, an inverted microscope setup and highly sensitive avalanche
photodiodes for detection in a confocal mode, and counting/TCSPC computer cards
to allow efficient data acquisition for further analysis. The TIR instrument also uses an inverted
microscope, but employs a high-sensitivity CCD camera for detection of multiple
single molecules in parallel.
We are currently focusing
on developing several key features of these methods.
1) The FRET methodology is
being optimized to detect fast structural transitions in proteins, using
continuous-flow mixing systems.
2) Techniques are being
developed to detect and utilize fluorescence lifetime information, using a
picosecond pulsed-laser system and time-correlated single photon counting
electronics.
3) Analysis methods are being
developed to evaluate additional molecular information from the detected
photons using brightness, polarization, time-correlations, and fluorescence
lifetime decay information. The information provided by these methods will
result in enhanced resolution of closely related subpopulations, and their
dynamics. One such example is shown in Figure 1 (12),
where the Y-axis represents the fluorescence lifetime of the donor fluorophore
and the X-axis shows the FRET efficiency computed using fluorescence
intensities of donor and acceptor, both measured from fluorescence bursts
emitted by individual donor-acceptor labeled DNA molecules. The Y-axis is
clearly observed to provide additional resolution of different subpopulations.
4) We have developed a
single molecule fluorescence quenching method that complements FRET by allowing
shorter distances and distance-changes to be measured in biological systems (7). In addition, multicolor FRET (9) and related methods are being developed to
allow simultaneous measurement of multiple distances or interactions in
molecules and complexes. These methods are initially tested using model DNA
molecules, and then applied to the study of folding, assembly and dynamics.
Group contacts: Edward
Lemke, Yann Gambin, Svitlana Berezhna
Biological
questions and systems.
Mechanisms
of Protein folding, Misfolding and Aggregation
Protein folding and protein-protein interactions.
We are generally
interested in exploring the distributions and dynamics of unfolded and partially
folded protein states. Besides their fundamental importance in protein dynamics
during folding and function, such protein states have also been implicated in
cellular functional mechanisms and also the pathogenesis of certain disease
states. Detailed observation of these dynamic protein states has been limited
so far by the averaging problems inherent in traditional experiments, and we
are applying single molecule techniques to overcome this obstacle. We have
previously shown that single molecule FRET experiments can be used to study the
simple two-state folding reaction of the protein Chymotrypsin Inhibitor 2, and
that several types of novel information about the folding reaction may be
recovered.(14) Figure 2 below shows FRET
histograms for the denaturation of Chymotrypsin Inhibitor 2 obtained using a
method we developed to make FRET measurements on freely diffusing molecules.
The histograms demonstrate the ability of the method to directly make observations
of different protein conformational states. The figure also displays
denaturation curves calculated from single-molecule data, along with curves
from ensemble measurements for comparison.
Having demonstrated the
feasibility of the single molecule FRET methodology to study folding, we are
now studying the more complex folding reactions of Barnase, and other proteins
to explore several unanswered questions in the area of protein folding. Both
the global folding/unfolding reaction and the concomitant changes in secondary
structure will be studied using single molecule fluorescence methods. We will
address several specific issues such as the determinants for the formation of
protein folding intermediates, how the folding landscape (conformational
heterogeneity of protein states, connectivity between them) changes with
sequence/environmental conditions, and how global and local protein structural
changes correlate during folding/unfolding transitions. In addition, the
influence of molecular crowding and molecular chaperones on the folding
reaction will be investigated, in order to provide a more physiological view of
the folding reaction.
Group Contacts: Allan
Ferreon, Samrat Mukhopadhyay
Collaborators: Schultz and Dawson groups (TSRI), and Weiss group (CI2, UCLA).
Protein misfolding and aggregation.
Protein misfolding and amyloid
formation have significant implications for human disease and biological
function. We are applying single molecule tools to study this process,
currently focusing on monomeric amyloid-forming proteins. In one example, we
have recently studied the yeast prion protein Sup35 using single molecule FRET,
coincidence and FCS methods (4). Our results
show that this protein, which acts as a non-DNA genetic element in yeast,
adopts a collapsed and rapidly fluctuating structure. We have also recently
completed a detailed ensemble thermodynamic characterization of the structural
properties synuclein, which is implicated in Parkinson’s and other
neurodegenerative diseases (3). We find that
this natively unfolded protein can adopt several structures under different SDS
and thermal conditions. This plasticity may play a key role in
synuclein’s function and in disease. In the future, we will extend our
single molecule studies to understand later stages of the amyloid formation
process.
Group
Contacts: Samrat Mukhopadhyay, Allan Ferreon
Collaborators: Susan Lindquist Lab (Whitehead
Institute)
RNA folding.
RNA folding shares many features
with protein folding, and this folding also represents a critical step in the
generation of functional RNA structures. Especially for larger RNA
molecules, slower folding has been observed and attributed to the formation of
misfolded structures. The hairpin ribozyme is a simple RNA structure that
surprisingly still shows relatively slow folding behavior. We are
studying this species using single molecule FRET in order to understand the
roles of loop, junction and sequence elements in its folding. We have
found that the four-way junction contributes uniquely to the stability of the
natural ribozyme, and that the folding of this natural form likely involves the
formation of an intermediate that is similar in structure to the native fully
docked ribozyme. The formation of such a "Quasi-docked"
intermediate helps stabilize the native structure by reducing the entropic cost
of its formation (10).
Group contacts: Ashok
Deniz
Collaborators: Millar Group
Assembly of complex biological
machines - Ribosomal fragments and subunits.
We are beginning to study
the detailed mechanisms of assembly of the bacterial ribosome using single
molecule methods. The small 30S subunit of the ribosome assembles from a
large RNA and 21 small proteins, through a complex process involving several
steps of binding and conformational changes. Our initial efforts focus on
the conformational properties and interactions of small RNA fragments from this
subunit with their protein partners. These studies are being extended to
the assembly of entire domains of the 30S subunit.
Group Contacts: Edward
Lemke, Yann Gambin
Collaborators: J. Williamson Group (TSRI)
Pathways and mechanisms of siRNA-induced RNAi
RNA
interference (RNAi) is a powerful biological process for specific silencing of
gene expression in eukaryotic cells and has remarkable potential for functional
genomics and drug discovery through in-vivo
target validation and development of novel gene-specific medicine. The RNAi
approach harnesses an endogenous cellular regulatory mechanism in which several
types of small RNA molecules (including microRNA and short interfering (siRNA))
bind to and mediate the destruction of specific mRNA molecules, preventing
their translation into proteins and inhibiting viral replication. In this
regard, siRNA can target molecules that conventional therapeutics find
difficult to reach, such as the Hepatitis C replication system. We are working on this and other systems
using an siRNA approach, primarily to understand the mechanistic features of
the siRNA-induced RNAi process.
RNAi is a complex cascade of events with multiple pathways, which in the most general case, regulates gene expression by inhibiting messenger RNA (mRNA) as recognized by complementary base pairing with small single-stranded RNA. However, before RNAi can discover its full potential as a therapeutic and investigative tool, several major problems have to be resolved. First, the precise mechanistic and functional features of the RNAi machinery are yet to be fully discovered and characterized. Second, in-vivo and intracellular delivery of siRNAs in a safe, targetable and controllable way remains a challenge. In our work, we are using advanced fluorescence microscopy techniques, such as multicolor confocal imaging, FRET and single particle tracking, to gain insights into these aspects of RNAi. For example, until recently, RNAi was thought to primarily operate in the cytoplasm of cells where mature mRNA is translated and key proteins of RNA induced silencing complexes (RISCs) were thought to localize and function. Recent work has uncovered the importance of RNAi in the nucleus, and we are now observing significant differences between the mechanistic pathways of small interfering siRNA that mediate cytoplasmic and nuclear-targeted RNAi (6). Additionally, several significant aspects of lipid-mediated delivery (lipofection) of exogenous silencing nucleic acids were revealed by a combined imaging approach with respect to improving down-regulation efficiency of lipofection technology (8). We are continuing to delve deeper into the complex and exciting world of RNAi mechanisms.
Group
contact: Svitlana Berezhna
Collaborators: Dr. Lubica
Supekova (supekova@scripps.edu) /
Peter Schultz Group
Last modified August 16, 2007. Please e-mail
comments/corrections to Deniz Lab Webpage
Administrator.