My Postdoctoral Work

Computational Studies of alpha-Helix Formation



Thanks for the image, Trevor ;-)

This work was done with Professor George Rose in the Department of Biochemistry and Molecular Biophysics , Washington University School of Medicine, St. Louis.

Along with Len Presta, George had shown that the side chains of residues near the termini of alpha-helices could form hydrogen bonds with the free backbone atoms in the first and last turns of the helix -- The "Helix Capping Hypothesis." It was proposed that these residues somehow specified the termination of the helix. Along with Reggie Aurora and Ed Harper, we set out to see if there were specific patterns, or motifs that could be used to predict the termination of alpha-helices. The short answer is - yes there are specific motifs that appear at the termini of helices. Whether they can be used to successfully predict the termination of helices is still to be determined. My work on capping motifs led to the following paper:


If you have Kinemage running on your computer, you can view a series of kinemages of capping boxes.


Current happenings in the Rose Lab

Structure/Folding Studies of the GroE Chaperonins



This image kindly provided by Helen Saibil

This work was done with Professor Paul Horowitz in the Department of Biochemistry at the University of Texas Health Science Center, San Antonio.

My work in Paul's lab involved structure/folding studies of the GroE chaperonins. These proteins from E. coli are thought to assist in the folding of proteins in vivo. My early work in the lab focused on the larger chaperonin, GroEL. Since GroEL binds to several unrelated proteins, it is believed that GroEL recognizes some non-specific feature of substrate proteins -- hydrophobic surfaces. In vitro the hydrophobic probe, bisANS is used to monitor the exposure of these hydrophobic surfaces on GroEL. However, this probe binds non-specifically. I developed a method for covalently incorporating bisANS into GroEL. I showed that bisANS binds to a region in GroEL that has been implicated by site-directed mutagenesis in substrate and GroES binding. In work that was done with Boris Gorovits, we showed that this same region of GroEL contains residual structure in the urea-denatured state of GroEL. This has implications for the folding of GroEL itself. This part of the work is represented by the following publications:


Since the rest of the chaperonin world seemed to be mostly ignoring GroES, I turned my attentions there once my work with GroEL ended. Since chaperonins can assist in the folding of other proteins, people have wondered what folds the chaperonins? Along with Boris and Jesse Ybarra, I showed that GroES could reversibly fold without other chaperonins. We also showed that GroES could assist the refolding of GroEL (which also turns out to be a hard protein to refold). This work has interesting implications in the stoichiometry of the chaperonin "machine" (2 GroES 7mers/1 GroEL 14mer). From this work, I then set out to elucidate the detereminants for GroES oligomerization in hopes of producing monomeric GroES for functional studies. I was able to show that the C-terminal 7 residues of GroES are required for oligomerization. Truncation of these residues by carboxypeptidase Y treatment produces monomeric GroES. This work has led to the following papers:


Once I had learned that the seven C-terminal residues of GroES were important for the oligomerization of the protein, I set out to find out the extent of the involvement of each of these residues in this interaction. With the help of John Chirgwin, we produced sequential deletion mutants of GroES up to and including seven residues. We found that deletion of the last 2 residues significantly destablised the oligomer such that at GroES concentrations used for experimental study, GroES existed as monomers. The most interesting aspect of this work is that these monomers possess all the activity normally associated with GroES function, i.e. ability to assist GroEL in refolding proteins and inhibition of GroEL ATPase activity. Isolation of GroES in which 3 or more C-terminal residues were removed was unsuccessful, in all likelihood due to the inherent instability of the monomeric protein in E. coli. This work led to the following paper:

A question that has emerged in my mind throughout my chaperonin work involves the stoichiometry of the groE operon and how that fits with the stoichiometry of the functional GroE machine. This question first arose from my work involving the refolding of GroES and GroEL (see above). During my characterization of the GroES deletion mutants, I had also observed that maximal chaperonin activity was always seen at a ratio of 2 GroES heptamers per GroEL tetradecamer. This stoichiometry in activity was confirmed using a direct binding assay developed with Boris that uses fluorescently labeled GroES to follow the real time binding of GroES to GroEL in solution. The idea that symmetric complexes are the functional chaperonin machine has also been proposed by other researchers such as Pierre Goloubinoff. At the end of my work in Paul's lab, I pushed this idea based on my work with GroES, and together with much work by Boris, we showed that symmetric complexes of GroES-GroEL-GroES can indeed be formed and that their formation is favored by and asymmetric distribution of ATP/ADP. This work was an appropriate and fitting end to my work in Paul's lab and resulted in the following co-first author publication:

You can also see some of my Analytical Ultracentrifugation work with GroES.


I have also created the Chaperonin Home Page .


Take a look at my CV .




Updated September 1, 1997



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