2008 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 protein misfolding diseases such as Huntington, Alzheimer, cancer and many others. Our approach is to use a hybrid of methods to characterize the biophysical and biochemical properties of chaperonins as well as develop engineering strategies to allow the design of chaperonins or substrates with new functionality.
We have assembled 12 independent investigators from 6 institutions (Baylor College of Medicine, Stanford University, University of California, San Francisco, Lawrence Berkeley National Laboratory, M.I.T. and M.D. Anderson Cancer Center) with different expertise to undertake this challenge. Our team members provide clearly defined complementary niches in the project and have assembled themselves into several subgroups to tackle various technical and biological fronts relevant to our overarching goals.
In the past three years, we have focused our efforts on refining various biophysical and computational tools to make them suitable for characterizing the chaperonin TRiC from mammalian sources and the related but simpler Mn-cpn from thermobacteria. Another important area of effort has been developing tools for engineering both the chaperonin and its substrates. Chaperonins are large oligomeric complexes that undergo an elaborate set of conformational changes during the reaction cycle that promotes polypeptide folding. The past year has brought us close to accomplishing our initial aims and anticipated milestones in the quantitative characterization of both chaperonins, which were not possible at the beginning of the project. More importantly, we are exploring various pathways to translational nanomedicine with clinical investigators beyond those already in our Center. Our latest accomplishments include:
- A preliminary 4.7 Å reconstruction has been obtained for Mm-cpn from single particle cryo-EM. This will provide the requisite structural framework for Mn-cpn chaperonin engineering and substrate-adaptor design.
- Two plausible structural models of TRiC with 8 non-identical subunits in two backto- back ring configurations have been derived from subnanometer resolution cryo-EM density map, chemical cross-linking data and model prediction. We are on the pathway to obtain the necessary structural information to re-design TRiC and its adaptors.
- The first de-novo backbone trace of bacterial GroEL derived from single particle cryo-EM was completed. This work represents a technological break-through in single particle cryo-EM for nanomachines. This structure reveals for the first time an asymmetric arrangement of the two rings in solution, which may represent a “primed conformation” of GroEL in the protein folding cycle.
- Our ABEL (Anti-Brownian ELectrophoretic) trap experiments on single chaperonins in solution show for the first time that TRiC can bind to different numbers of ATP molecules, yet still shows the average cooperativity observed in bulk experiments. These measurements demonstrate the heterogeneous structural complexity of the chaperonin in a nucleotide bound state. This observation raises the question as what will be the optimal number of bound ATP to obtain efficient folding. This is an important factor to be considered in the design of new and more efficient TRiC.
- Single-molecule FRET experiments show for the first time that selected regions in von-Hippel-Lindau (VHL), a tumor suppressor protein can both contract and expand during folding inside the reaction chamber of the bacterial GroEL chaperonin. This methodology opens up many future experiments studying our model chaperonins: TRiC and Mm-cpn in interaction with targeted substrates related to known diseases. The dynamic motions of substrates are important factors to consider in the design of an effective chaperonin.
- Computer simulations including solvent inside the chaperonin reaction chamber have suggested ways to design novel GroEL mutants with increased refolding ability. The accuracy of in silico prediction of the chaperonin activity as a function of the chaperonin structure can play an important role in the engineering phase of our research.
- Our first in silico design of an adaptor between VHL and TRiC is now validated by experimental results which suggest optimism in our proposed approach for engineering nanomachines with new functionality.
- Several new computational tools are being developed: to determine structures of conformationally variable regions of the chaperonins from cryo-EM images; to identify regions of conserved structural motifs in a cryo-EM map of nanomachine; to fit models to cryo-EM densities of nanomachines with varying conformations at different physiological states; to derive low resolution models of nanomachines in different chemical states from solution x-ray scattering.
- A number of new projects were initiated as a result of the supplemental funds including the biophysical characterization of Huntingtin fibril aggregates by singlemolecule imaging and electron cryo-tomography; expanding the computational library of potential candidates of adaptors for chaperonins and inhibitors for Htt diseases; and purifying chaperonin from human cell lines.
- Eric Jonasch, a clinical investigator, has begun his active participation in screening and designing possible adaptors for the mutants of von Hippel Lindau cancer suppressor protein which fail to interact with the TRiC.
- We successfully conducted a workshop in January 2008 on translational nanomedicine with 10 clinical investigators from various academic institutions to brainstorm for appropriate clinical problems that can be benefited by our NDC expertise. We have continued a monthly online tele-seminar on protein folding machinery via Webex protocol.
- Our annual summer undergraduate research program continued for a second year in several different institutions.
- We conducted a productive one-day workshop with the NSF-funded Center for Biological and Environmental Nanoscience to discuss the grand challenges in nanomedicine from the respective points of views of biomedical scientists and nano-scientists.
- A symposium highlighting the research of several of our NDC members was organized for the 2007 annual meeting of Protein Society.
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