Our laboratory explores the folding, structure, function
dynamics of proteins. Most of our work is based on the use of electrospray
mass spectrometry (ESI-MS). Researchers working in our laboratory
also have access to a wide range of other biophysical techniques,
including high-field NMR, fluorescence, circular dichroism, UV-VIS, and
stopped-flow spectroscopy, differential scanning calorimetry (DSC),
analytical ultracentrifugation, surface-plasmon resonance (Biacore),
and many others. A nanofabrication facility for the development of
microfluidic devices is also available.
Current research in our group includes the following
Protein Folding Kinetics and Mechanisms
The native state of a protein in solution is characterized by
a tightly folded and highly specific three-dimensional
structure. This native conformation is determined by the linear
sequence of amino acids along the polypeptide backbone. In high
concentrations of denaturants such as acid, proteins adopt a largely
disordered (unfolded) conformation. Unfolding is
reversible, i.e., the protein
spontaneously refolds into its native
structure when the denaturant is removed. This process occurs on the
time scale of milliseconds to minutes.
Protein folding is a remarkable
phenomenon; there are billions of different possible ways to fold a
protein chain and yet every protein "knows" how to find its native
structure. Sometimes short-lived intermediates become populated
during folding. The exact role of these intermediates for the overall
folding mechanism is still a matter of debate. However, it is clear
that the identification and structural characterization of these
species is an important step towards a better understanding of protein
Electrospray ionization mass spectrometry (ESI-MS) is
one of the main techniques that is used in our lab. ESI generates
multiply protonated protein ions in the gas phase. "Time-resolved"
ESI-MS is a technique that allows the
folding kinetics and mechanisms to be studied in great detail.
Time-resolved ESI-MS can be used in conjunction with hydrogen/deuterium
exchange (HDX), thus
allowing the detailed characterization of folding intermediates. We
pursue both top-down and bottom-up MS approaches. Complementary
information can be obtained by laser-induced
1: Time-resolved ESI-MS experiments provide "molecular
movies" that reveal how a protein folds. In this OH radical labeling
data set unfolded regions are shown in red, native elements are
depicted in blue.
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One of our goals is to study the refolding and assembly
of protein-protein complexes.
Very little is known about the mechanisms by which these complexes are
formed, bind their prosthetic groups, and eventually adopt their
biologically active conformations.
2. In this experiment we used millisecond time-resolved
measurements to decipher the mechanism of a coupled folding/binding
reaction. The side chain solvent exposure ranges from high (red) to
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Protein Conformational Dynamics
Proteins are highly dynamic systems that undergo
conformational fluctuations and unfolding/refolding transitions on
various time scales. In many cases, these conformational dynamics are
crucial for the biological function of a protein. Time-resolved
ESI-MS used in conjunction with HDX is a powerful tool for
monitoring these dynamics (Figure 3). We are studying the
behavior of proteins under conditions that lead to amyloid and other
forms of aggregates. This work has direct implications for
understanding the disease mechanisms of Alzheimer's and a
number of other disorders. The combination of top-down electron capture dissociation
(ECD) with HDX has opened up
new avenues for studying protein folding and dynamics.
Figure 3. Conformational
dynamics of apo-myoglobin probed by top-down ECD HDX/MS. Some regions
of the protein are highly dynamic (red), others show intermediate
dynamics (green), whereas a number of elements are quite rigid (blue).
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Noncovalent Ligand-Protein Interactions
Drug discovery and development relies on the
identification of high-affinity ligands to specific target proteins.
Our group has developed a novel ESI-MS-based approach for
this purpose. Potential ligands are usually relatively small compounds
that undergo rapid translational diffusion as long as they are free in
solution. However, diffusion is drastically slowed down for ligands
that are bound to a protein (Figure 4). We have devised a way of
measuring the diffusion coefficients of a large number of potential
ligands simultaneously by ESI-MS. It appears that this method could
become an important tool for the high throughput screening of
compound libraries. This approach is of significant interest to the pharmaceutical
4. A small molecule that is free in solution diffuses
rapidly (left). Diffusion is drastically slowed down upon noncovalent
binding to a protein (right).
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Development of Rapid-Mixing Systems for Kinetic Studies on
ESI-MS has enormous potential for kinetic studies on a wide
range of biochemical systems. Our laboratory is one of the few places
worldwide where on-line MS-based kinetic studies can be carried out
in the millisecond regime. We have developed a number of novel
approaches in this area (Figure 5), and we are currently
the use of microfluidic systems to improve the time resolution
of these techniques down to the sub-millisecond time range.
5. Stopped-flow ESI-MS and continuous-flow rapid mixing
ESI-MS are two techniques for kinetic experiments that have been
developed in our laboratory.
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Enzyme Kinetics and Mechanisms
Immediately after initiating an enzymatic reaction there is a
short time range (usually some milliseconds to seconds, depending on
the rate constants involved) during which the system approaches
steady-state conditions. The mechanism of the reaction dictates the
order by which short-lived intermediates become populated
successively during this period. Individual rate constants can be
measured from which the energy barriers along the reaction coordinate
can be estimated. Due to its high selectivity and sensitivity, time-resolved
ESI MS is a powerful tool for kinetic studies in this pre-steady-state
regime. In addition, we are interested in exploring the role of
enzyme conformational fluctuations
for the mechanism of catalysis.
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Fundamental Aspects of Electrospray Ionization
Despite the widespread use of ESI-MS, the exact nature of the
processes leading to the formation of gas phase ions from molecules in
solution is still not well understood. We are in the process of
the physical reasons underlying the relationship between protein
conformations and ESI charge state distributions. Molecular dynamics (MD) simulations
turned out to be a key tool for gaining insights into the ESI
processes occurring during ESI represent another focus of our work.
Figure 6. This frame is part of an MD trajectory
that shows how an ion is ejected from a highly charged ESI droplet.
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