Dr. Carl Frieden

Department of Biochemistry and Molecular Biophysics

Biochemistry Program
Molecular Biophysics Program
Molecular Cell Biology Program

Current Lab Members and Collaborators:

Dr. Carl Frieden, Dr. Melissa Brereton, Berevan Baban,
Dr. Linda Kurz and Dr. George Drysdale.


Biochem 5312

There are two data sets that should be downloaded. Click Here
Problem Set Answers

Biochem 5325 - Lectures notes

Overview of Research Projects

Current Projects

ApoE proteins and Alzheimer's Disease

While apolipoproteins are essential for lipid and cholesterol metabolism one such protein in the brain, called ApoE4 is the major risk factor for late-onset (onset after age 60), Alzheimer's disease (AD). The apoE4 allele is present in about 10-15% of the population and half the individuals with two copies of apoE4 protein will develop AD. ApoE3, the common form of apoE, differs from apoE4 by a just a single amino acid (out of 299) but is benign with respect to developing AD. Thus, the two proteins are functionally different. We are examining, using biophysical and other techniques, how this single amino acid change can give rise to these functional differences.

Amyloid beta, , is a small peptide associated with plaques in those who have Alzheimer's Disease. Genetic, biochemical, and animal model studies strongly suggest that apoE4 is likely to influence Alzheimer's Disease pathogenesis via effects on the metabolism of the 38-43 amino acid amyloid-β (Aβ) peptide.

Curli proteins. Bacterial communities, called biofilms, are important in various types of infections, including urinary tract infections (UTIs), chronic skin wounds, otitis media and lung infections in cystic fibrosis patients. For E. coli and other Enterobacteriaceae biofilms, adhesive amyloid fibers called curli can be a major proteinaceous constituent of the extracellular matrix. Curli promote biotic and abiotic surface colonization, stabilize cell-cell contacts allowing cell aggregation and thickening of the biofilm layer, and confer resistance to the biofilm against environmental stresses and biocides. The structural subunits of curli amyloid fibers are CsgA (major component) and CsgB (minor component), CsgE and CsgF which may serve a chaperone function and CsgG, a lipoprotein that localizes to the outer membrane as an oligomeric pore structure and is required for the export of curli subunits to the cell surface. Both CsgA and CsgB are intrinsically disordered proteins (IDPs). In vitro, CsgA and CsgB can self-associate to form high molecular weight aggregates/fibrils. We are studying the properties of the curli subunits and their mechanism of aggregation.

Click here to see recent publications.

For a List of References on These and Other Projects, Click Here

Overview of Previous Projects

Protein folding, protein dynamics, protein structure/function relationships, protein-protein interactions and polymerization/aggregation mechanisms are projects that we have studied in this laboratory.

Protein Folding

The long term goal of the protein folding studies was 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 work with the apoE family of proteins, with the role of proline in protein folding and with intrinsically disordered proteins such as amyloid beta and the bacterial protein CsgA.

Many of the studies involved 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. A website for fluorine chemical shifts has been established at

Intrinsically Disordered Proteins

Similar to the project with curli proteins we have studied the dynamics and aggregation properties of intrinsically disordered proteins.

We use real-time NMR measurements of fluorine labeled proteins to measure the rates of protein folding and unfolding. Most studies involve the incorporation of fluorine labeled tryptophan, phenylalanine, tyrosine or proline. The role of proline in protein folding. We are incorporation fluoroproline into RNase T1 and barstar and following the cis to trans isomerization by 19F-NMR in order to determine the role of proline in the protein folding and unfolding process in systems that contain multiple proline residues.

Specific information about some of these projects is given below.

Past Projects

groel actin.gif papd.gif dhfr





ifabp ada.gif barstar.jpg





Interactions of the Molecular Chaperonin GroEL with Dihydrofolate Reductase

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.


Folding Studies of PapD

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


Folding and Polymerization of Actin

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.


Real-time Folding Studies of E. coli DHFR using 19F Stopped-flow NMR

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.


Folding Studies of Intestinal Fatty Acid Binding Protein

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.


Folding of Adenosine Deaminase

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

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: https://biochem.wustl.edu/faculty/frieden
last updated: 4/3/18