since 3/97
Biochemistry Program
Molecular Biophysics Program
Molecular Cell Biology Program
| Dr. Carl Frieden, Scott Crick, Dr. Greg DeKoster, Dr. Kanchan Garai, Dr. Sourajit Mustafi, Dr. Sudha Cowsik, Berevan Baban, Dr. Linda Kurz and Dr. George Drysdale. |
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You can download the newest versions of Kinsim and Fitsim for the PC
from this site. Click Here to
view the release notes and download the compressed file. You will also
find a link to a short help page.
NOW YOU CAN RUN KINSIM AND FITSIM ON YOUR MAC! Requires downloading a DOS emulator like DOSBOX (which is free). Click here for instructions. Instructions using DOSBOX were written by Scott Crick. |
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Biochem 5312 There are two data sets that should be downloaded. Click Here Problem Set Answers
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| Biochem 5325 - Lectures notes |
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IFABP |
ADA |
Barstar |
CsgA |
Intrinsically
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Protein folding, protein dynamics, protein structure/function
relationships, protein-protein interactions and polymerization/aggregation
mechanisms are projects currently under study in this laboratory. We are
particularly interested in measuring the kinetics of side chain packing
and stabilization during folding as well as dynamics in the unfolded
state.
The long term goal of the protein folding studies is to understand the
nature of the unfolded and intermediate structures on the unfolding and
refolding pathways, including the role of proteins that assist folding
(called chaperonins). The work uses site-directed mutagenesis and
techniques such as 19F and proton NMR, circular dichroism,
fluorescence measurements and x-ray crystallography. Current studies include the
intestinal fatty acid binding protein, barstar, the Trp cage, adenosine
deaminase and some amyloid forming proteins.
Many of the studies involve incorporating fluorine labeled amino acids
into the protein and then examining the NMR spectrum. We use stopped flow
methods in conjunction with a fluorine cryoprobe for these measurements.
From data collected in current and past projects we have a large database
of fluorine chemical shifts in proteins. These shifts are sensitive to low
concentrations of denaturant, to temperature and to pH. In collaboration
with computational faculty, we are attempting to explain how these shifts
may mirror structural or conformational changes in proteins. A website for
fluorine chemical shifts has been established at
http://biochem.wustl.edu/~bmbnmr/Fluorine.html.
Proline isomerization between the cis and trans forms is believed
to be
responsible for the slow rate of folding of many proteins. For those
proteins that have multiple proline residues, it is currently not possible
to measure the rate of isomerization of each proline during folding. To
solve this problem, we are incorporating 3-19F-proline into the
protein.
As with the incorporation of other fluorine labeled amino acids (see other
projects), each proline residue in the native protein appears to have a
specific resonance peak. Using stopped-flow NMR experiments with the
3-19F-proline, we can measure the rate of proline isomerization
for each proline residue and thereby define which proline(s) are responsible for
the slow rate of folding.
Amyloid formation:
CsgA is a 14 kD bacterial protein that aggregates to form amyloid fibers. The monomer in solution appears to be unstructured. Aggregation in enhanced by the presence of another protein called CsgB. We are examining the characteristics of the monomer and of the aggregation process. We are studying the formation of amyloid fibrils from AB and the nature of polyglutamine tracts alone and in the huntingtin protein. Of particular interest is the dynamic motions within the proteins as examined by fluroescence methods such as Fluorescence Correlation Spectroscopy (FCS) Fluorescence and Conformational Dynamics: Fluorescence Correlation Spectroscopy (FCS) is a method capable of measuring diffusion coefficients as well as the rate of conformational changes in the microsecond range. We have been using this method to measure these rates in native as well as unfolded or intrinsically disordered proteins. Specific information about some of these projects is given below. |
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The goal of this project is to determine the mechanism of folding of the
intestinal fatty acid binding protein (IFABP). This
protein is one of a family of proteins that bind fatty acids, bile salts
and retinoids. This family is primarily beta-sheet with
a large central cavity into which the ligand binds. Cysteine-
and proline-free, IFABP is a model protein for studying the
mechanism of folding.The lack ofproline allows us to explore the role of
proline in the folding process. We have developed the technique of turn
scanning (mutagenesis in turns) to examine the role of turns in the folding
process. By NMR, we are currently examining structrual changes that occur
throughout the molecule as a consequence of single site mutations. We are
also using this protein to explore the method of fluorescence correlation
spectroscopy (FCS) in combination with fluorescence resonance energy
transfer (FRET) as it applies to protein structural changes in both the
native and unfolded states. To read recent abstracts of this work, click Here. |
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Work with the murine (mouse) adenosine deaminase continues our attempt to
understand the folding of larger and larger proteins. Adenosine deaminase
has a molecular weight of 40 kD and is present in virtually all mammalian
cells. It is a key enzyme in purine metabolism. Lack of enzymatic
activity, leading to severe T, NK and B lymphocytopenia, is associated
with about 20-30 percent of children with severe combined immunodeficiency
(SCID). In addition it contains a tightly bound Zn atom that is essential
for enzymatic activity. There are numerous reports in the literature of
mutations that lead to the loss of enzymatic activity associated with
SCID. Some, understandably, are in the active site of the enzyme while
others, surprisingly, are distant from the active site. We will study why
distant mutations lead to partial or total loss of enzymatic activity. We
suggest that a possible explanation for the loss of activity in these
distant mutations arises either from large conformational changes or from
a loss of ability of the protein to fold properly. The studies involve
expression of wild-type and mutant murine adenosine deaminases (the murine
enzyme is highly homologous to the human enzyme) followed by
characterization of the properties using fluorescence, circular dichroism
and NMR techniques as well as enzymatic activity. In particular we are
interested in the characterization of folding properties and the role of
Zn in folding.
To read recent abstracts of this work, click Here
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Intrinsically Disordered Proteins |
| We are studying the dynamics and aggregation properties of intrinsically disordered proteins including AB, polyglutamine (alone and in huntingtin Exon1) and CsgA. CsgA is a bacterial protein secreted into the media forming fibrils that surround the bacteria. Studies include the behaviour of proteins unfolded by denaturant. The main methods for these sutdies include FCS and NMR (after incorporation of fluorine labeled amino acids). |
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GroEL |
Actin |
PapD |
DHFR |
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The goal of this work is to understand the GroEL-mediated
folding mechanism. GroEL
is a member of the Hsp60 class of chaperones,
which are tetradecamers of identical 57.2 kDa monomers. The chaperonin
binds unfolded proteins and generally increases the final yield of native
protein without increasing the rate of folding. The formation of stable
complexes with GroEL is most often discussed, and while, for example,
murine dihydrofolate reductase (MuDHFR) does form a stable complex, the
complex formed with the structurally homologous E. coli DHFR
(EcDHFR) is transient (at 22 degrees C). For both DHFRs, the concentration
of bound
protein increases as the temperature is increased. Using a variety of
biophysical approaches, we are examining the
kinetic mechanism of folding as well as the thermodynamic parameters for
the binding of these DHFRs with GroEL. Our most recent work examines
the role of ligands such as K+, Mg2+, ATP
and GroES on the folding mechanism. To read recent abstracts of this work, click Here. |
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PapD is a protein required for the formation of pili in pathogenic
bacteria. It, however, does not get incorporated into the pilus and is
believed to function as a chaperone for the folding of other proteins
which make up the pilus structure. This protein is rich in proline and has
two distinct domains. The folding is sufficiently slow that we can apply
NMR techniques to examine the folding process much like have been used
with dihydrofolate reductase (see above). Currently we are examining the
role of domain-domain interaction on the folding process.
To read recent abstracts of this work, click Here |
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The goal of this work is to understand the molecular mechanism of actin
polymerization and filament function. We are investigating the role of
specific amino acid residues in the mechanism by comparing the kinetics of
self- polymerization of mutant actin proteins with that of wild type, in
the presence and absence of various actin binding proteins. Because yeast
genes are more readily manipulated than those of higher eucaryotes, we are
primarily studying yeast actin including several actin mutations that have
been phenotypically characterized in nematodes which we have put into
yeast actin.
To read recent abstracts of this work, click Here. |
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| In this work, we are studying sidechain environment and behavior during the refolding of E. coli dihydrofolate reductase (DHFR) in real time by stopped-flow NMR techniques. E. coli dihydrofolate reductase has five tryptophans which have been replaced with 6-19F-tryptophan. The resonances assigned to each tryptophan are resolved in both unfolded and native DHFR, allowing us to monitor the environment of individual tryptophans during unfolding and refolding in real time using stopped-flow 19F NMR techniques. This allows, for the first time, measurements of stabilization of specific regions of a protein during the refolding process either in the presence or absence of ligands. The work is being extended using 19F-labeled phenylalanine as markers for specific region or domain stabilization during refolding. The refolding and unfolding kinetics have also been examined by stopped-flow circular dichroism and stopped-flow fluorescence techniques and compared to the sidechain environment observed by stopped-flow 19F NMR. To read recent abstracts of this work click Here. |
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Dr. Carl Frieden Department of Biochemistry and Molecular Biophysics, Box 8231 Washington University School of Medicine 660 South Euclid St. Louis, MO 63110 (USA) office: 314-362-3344 lab: 314-362-3342 or -3359 FAX: 314-362-7183 send mail to frieden@biochem.wustl.edu
URL: http://biochem.wustl.edu/cflab |