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PROTEIN-MEDIATED PROTEIN FOLDING 
Arthur L. Horwich, M.D., Associate Investigator 
Chaperonins 
A major portion of the research in Dr. Horwich's 
laboratory is aimed at understanding how proteins 
acquire their functional structures in the living cell. 
Newly translated proteins emerging from ribosomes 
and proteins translocated through membranes must 
be folded into the correct tertiary structures to 
achieve biological activity. For many years this was 
believed to be a spontaneous process, but recent 
studies indicate that special protein components 
called molecular chaperones mediate folding in 
vivo. Dr. Horwich's laboratory uncovered such a 
component in mitochondria: heat-shock protein 
60 (hsp60). It was the component affected in a 
temperature-sensitive lethal mutant of yeast in 
which proteins imponed into the mitochondrial 
matrix failed to be folded and assembled into active 
forms. The misfolded proteins were found as insolu- 
ble aggregates. 
Residing in the mitochondrial matrix as a homo- 
oligomeric l4mer complex, hsp60 is composed of 
two stacked rings, each containing seven radially 
arranged 60-kDa monomers. It binds newly im- 
ported mitochondrial proteins in unfolded forms 
and mediates folding in a process requiring ATP and 
a second cooperating component. It shares 60% of 
its amino acids and the same quaternary structure 
with both a protein of the bacterial cytoplasm, 
groEL, and the RUBISCO-binding protein of chlo- 
roplasts. The three related components, termed 
chaperonins, are probably evolutionarily related 
through events of endosymbiosis from which mito- 
chondria and chloroplasts emerged. 
Chaperonin Mechanism 
In vitro studies with groEL have reconstituted the 
native active form of several monomeric proteins 
diluted from denaturant. In studies carried out in 
collaboration with Dr. Ulrich Hartl and his co- 
workers, groEL-mediated folding was dissected into 
two steps: 1) binding of an unfolded polypeptide, 
with a stoichiometry of one or two molecules per 
groEL 1 4mer complex, with stabilization in the con- 
formation of an early-folding intermediate called 
"molten globule," containing a native-like second- 
ary structure, a nonorganized tertiary structure, and 
solvent-accessible hydrophobic residues; and 2) 
folding, mediated upon addition of MgATP and the 
cooperating component groES, a seven-member ring 
composed of 10-kDa monomers, which binds with a 
stoichiometry of one groES ring per groEL l4mer. 
Folding is associated with ATP hydrolysis (~100 
ATP/monomer), presumed to be expended in rear- 
rangement of the groEL structure. The presence of 
groES appeared to couple the substrate polypeptide 
to groEL, potentially enabling its stepwise folding 
and release. 
Chaperonin-like Heat-Shock Protein 
in Thermophilic Archaebacteria 
and a Relative in the Eukaryotic Cytosol 
The mechanism of chaperonin-mediated folding 
may not be confined to the compartments in which 
groEL, hsp60, and RUBISCO-binding protein are lo- 
calized. The major heat-shock protein of thermo- 
philic archaebacteria was discovered to exhibit a 
similar quaternary structure and functional features. 
202 
