Protein Design, Protein Recognition and Molecular Chaperones
EMSL Project ID
2140
Abstract
Molecular chaperones such as heat shock proteins assist in protein folding, import, translocation and degradation of proteins. A new protein called BAG1 binds to and modulates the chaperone activity of Hsc70. BAG proteins also and may modulate activity of nuclear hormone receptors by interaction with Hsc70/Hsp70. In human tumor cells lines, levels of BAG-1L are elevated in hormone dependent cancers such as prostate, breast and lymphoid malignancies. BAG1 may be a link between androgen receptor or estrogen receptor and heat shock chaperones. It has been suggested that BAG proteins and the cell death pathway could be new drug targets for cancer patients who are no longer responding to endocrine therapy. From earlier homology modeling of BAG1/Hsc70 we identified a binding crevice on Hsc70 as the site for BAG1 interactions and confirmed the prediction with chemical shift experiments followed by mutagenesis. From these results, a 'mini'domain of the Hsc70 ATPase domain has been produced by recombinant DNA methods that contains the interaction sites in BAG recognition. This fragment is 60% helical. The plan is to determine the structure of the 'mini' domain by NMR methods and to determine the structure of the 'mimi'domain in complex with BAG1. NMR experiments will be carried out with one partner unlabeled and the other partner labeled. The strategy is to use 15N-filtered and/or 13C,15N-filtered experiments. Our experience with BAG1 protein which is highly helical demonstrates that high field instruments are required to resolve spectral ambiguities. We were awarded time on the 600 and 750 MHz spectrometers for the last cycle for this project, however our scheduled time has not yet come to the top of the queue. Nevertheless, from our experience with macromolecular NMR, for the coming cycle we anticipate that time will be also be needed to complete the study, and therefore we request time on the 600MHz and 750 MHz spectrometers.
Project Details
Project type
Capability Research
Start Date
2001-07-25
End Date
2001-10-02
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members