The human mitochondrial Hsp60 in the APO conformation forms a stable tetradecameric complex |
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Authors: | Adrian S. Enriquez Humberto M. Rojo Jay M. Bhatt Sudheer K. Molugu Zacariah L. Hildenbrand |
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Affiliation: | 1. Department of Chemistry, The University of Texas at El Paso, El Paso, TX, USA;2. Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA;3. Inform Environmental, Dallas, TX, USA |
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Abstract: | The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding of mitochondrial proteins and is of vital importance to all cells. This chaperonin is composed of 2 distinct proteins, Hsp60 and Hsp10, that assemble into large oligomeric complexes that mediate the folding of non-native polypeptides in an ATP dependent manner. Here, we report the bacterial expression and purification of fully assembled human Hsp60 and Hsp10 recombinant proteins and that Hsp60 forms a stable tetradecameric double-ring conformation in the absence of co-chaperonin and nucleotide. Evidence of the stable double-ring conformation is illustrated by the 15 Å resolution electron microscopy reconstruction presented here. Furthermore, our biochemical analyses reveal that the presence of a non-native substrate initiates ATP-hydrolysis within the Hsp60/10 chaperonin to commence protein folding. Collectively, these data provide insight into the architecture of the intermediates used by the human mitochondrial chaperonin along its protein folding pathway and lay a foundation for subsequent high resolution structural investigations into the conformational changes of the mitochondrial chaperonin. |
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Keywords: | Human mitochondrial chaperonin negative stain transmission electron microscopy |
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