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Purification and Characterization of the Cytosolic TRiC Complex from HeLa Cells
Author(s) -
Knee Kelly Marie,
Goulet Daniel Robert,
Jameel Shea,
King Jonathan A
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.672.5
Subject(s) - chaperonin , hela , crystallin , cytosol , folding (dsp implementation) , actin , biochemistry , biology , protein folding , chemistry , biophysics , microbiology and biotechnology , cell , enzyme , electrical engineering , engineering
The group II chaperonin TRiC is required for actin and tubulin folding and is thus essential for the reproduction of all eukaryotic cells. We have purified Human TRiC from cervical adenocarcinoma (HeLa) cells. To assess the substrate‐binding properties of Human TriC, we are characterizing its interactions with human γCrystallins. The γCrystallins are a family of β‐sheet eye lens proteins, whose partially folded intermediates have been well‐characterized. The Archaeal group II chaperonin Mm‐Cpn, from M. marapaludis, is a homolog of human TriC, and it has been shown that the complex suppresses aggregation of partially folded Human γCrystallins, and refolds the Crystallins to a native‐like conformation. We are investigating the potential of Human TRiC to suppress aggregation and restore unfolded γCrystallin to a native fold in a similar manner to Mm‐Cpn, using UV/Vis spectroscopy, and size exclusion chromatography. In collaboration with Wah Chiu (Baylor College of Medicine) and Judith Frydman (Stanford University) of the Center for Protein Folding Machinery, we are attempting to visualize the crystallin substrate in the chaperonin/substrate complex by Cryo‐EM. Supported by an NIH Roadmap Award.