2009 Progress Report Executive Summary
The ultimate goal of our NDC is to engineer chaperonins with new functional properties, and/or substrate adaptor molecules, to prevent aggregation and/or refold proteins responsible for misfoldedprotein diseases. Our approach is to develop and integrate methods to characterize the biophysical and biochemical properties of chaperonins, as well as to develop engineering strategies for designing chaperonins or substrates with new functionalities.
With the financial supplement and scale-up support, we have recruited four new clinical investigators from Baylor College of Medicine and the Stanford Medical School to join our NDC. These new team investigators have led us down an exciting pathway towards translational medicine. Each of our team members provides clearly defined complementary project niches and has assembled into several sub-groups to tackle our goal-relevant technical and biological fronts.
In the past year, we have made substantial progress in several experimental and computational methods for characterizing chaperonins and nanomachines in general. These include single particle cryo-EM at near atomic resolution, single molecule imaging to measure chemical kinetics, modeling based on low and medium resolution structures of highly flexible nanomachine made up of multiple molecular components, and simulation of chaperonin dynamics in the solvated state. All these advances are critical to characterize the biophysical properties and functional activities of chaperonins and their substrates. All of these accomplishments meet our original milestones. Some of the developments are beyond our original anticipations. Our clinical investigators are progressing steadily and methodologically in collaboration with our basic science investigators. Our major accomplishments include:
- A 4.3 cryo-EM reconstruction of wild type Mm-cpn in the ATP-induced state. This is the highest resolution structure determination of any biological nanomachine ever reported without a crystal structure of its components or domains. Its map is clear enough to allow a C-? backbone trace and identifying sidechains and nucleotide density. An accompanying 8 cryo- EM study of the mutant Mm-cpn with deletion of the protruding lid without the nucleotide allows us to propose a structural mechanism describing how Mm-cpn closes its lid upon ATP hydrolysis. Key residues are identified to make such a large mechanical motion necessary for triggering protein folding. These structural details are feeding into our pipeline for engineering new Mm-cpns with different biochemical characteristics.
- X-ray crystallography is making substantial progress in obtaining crystal structures of mutant Mm-cpn in apo and nucleotide induced states. The crystal quality continues to improve. The eventual crystal structures will be used together with cryo-EM models for designing adaptor molecules to facilitate folding of target substrates.
- The single molecule imaging team has overcome technical difficulties in making appropriately labeled ATP to allow measurement of number of ATP bound to a single TRiC. Such single molecule measurement is important to characterize the biochemical reaction of chaperonin where ATP hydrolysis is critical. It has applicability to many other nanomachines requiring ATP.
- The discovery that TRiC can rescue Huntingtin fibril aggregation has opened up much computational and structural research initiatives. Computational modeling is shown to predict conformational behavior of Htt correctly according to its consistency with experimental data. Such prediction accuracy will facilitate future adaptor design. Cryo-EM performed in the Chiu lab of the Htt fibril with TRiC suggests an intriguing mechanism of TRiC capping the tip of the fibril.
- A new computational method is being developed, tested and validated for its sub-angstrom accuracy in predicting conformation of looping region of protein, which is often a functional region for interacting other cellular components.
- Various methodology innovations have been made to refine cryo-EM structures, to segment domains and components of cryo-EM maps and to fit cryo-EM maps with PDB models at various resolutions. These technological advances will be deployed in the future cryo-EM image analysis of chaperonins with substrates at various folding stages.
- Several newly initiated projects under the translational medicine have begun with steady progress. These projects are relevant to misfolded or protein aggregation resulting in a variety of diseases: cystic fibrosis, von Hippel Lindau disease, Down syndrome, Alzheimers disease, apoptotic cell death and cataracts.
- Armed with the computational prediction, adaptor molecules are being proposed and tested with a cellular screening assay for A-beta-involved disease.
- We have initiated an unexpected collaboration with a clinician at UC-Irvine to investigate whether chaperonin derivatives can be introduced to the mouse brain to reduce aggregation toxicity.
- Experiments are being set up to use C. elegans as a model for testing the feasibility of adaptormediated rescue of the A-beta peptide toxicity.
- Collaborations are being carried out with other NDCs (Mike Sheetz, Wendel Lim, and Ehud Isacoff) and the Roadmap on Computational Biology to leverage our complementary purposes to pursue our respective goals.
- We sponsored two workshops successfully on cryo-EM image processing and modeling nanomachines at different resolutions. Attendance was 130 and 30 for these workshops, respectively.
- Our summer undergraduate research program was well executed with impressive virtual presentations by the trainees in the fall of 2008.
- Our regular monthly tele-seminars have been well attended. An impressive list of world-class leaders in the protein-folding field is scheduled to speak in the spring of 2009.
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