Protein Folding in Vivo 
Arthur L. Harwich, M.D. — Associate Investigator 
Dr. Norwich is also Associate Professor of Human Genetics and Pediatrics at Yale University School of 
Medicine. He received A.B. and M.D. degrees in biomedical sciences from Brown University. His internship 
and residency training in pediatrics were done at Yale. His postdoctoral research training was at the Salk 
Institute with Walter Eckhart and at Yale University with Leon Rosenberg. 
UNTIL recently it has been assumed that 
newly made proteins in the living cell, com- 
prising amino acid chains with characteristic se- 
quences, are able to fold spontaneously into pre- 
cise three-dimensional structures that exhibit 
biological activity. Such folding has been ob- 
served in test-tube experiments, where many 
proteins unfolded by denaturing agents can be 
diluted from these agents and observed to refold 
into their biologically active forms. Recent stud- 
ies, however, suggest that in the cell the process 
of folding is assisted by other specialized pro- 
teins. We originally identified such a protein 
while studying mitochondria, the intracellular 
organelles that carry out energy metabolism. 
Most of the proteins of mitochondria are first 
made outside the organelles, in the cytosol, and 
then imported through two membranes to reach 
the innermost mitochondrial "matrix" compart- 
ment. To traverse the membranes, the newly 
made proteins are first unfolded on the cytosolic 
side. After import, the proteins refold on the in- 
side of the organelles into their biologically ac- 
tive conformations. We identified a mutant cell 
in which mitochondrial proteins were imported 
but failed to fold into biologically active forms. 
The mutation was found to affect a protein that 
normally resides in the matrix compartment, 
called heat-shock protein 60 (hsp60). 
This protein was originally identified by the 
observation that its abundance is increased about 
twofold in response to incubation of cells at high 
temperatures. However, it is an abundant protein 
even before heat shock, and our genetic analysis 
demonstrated that, consistent with a critical base- 
line function, hsp60 is required not only at high 
temperatures but at all temperatures. The in- 
creased level produced in response to heat stress 
could represent an effort to try to refold effi- 
ciently mitochondrial proteins that heat has 
denatured. 
In the mitochondrial matrix, hsp60 is found in 
a higher order structure, a complex. Fourteen 
copies of the protein are arranged in two stacked 
rings, a "double donut . ' ' Each ring contains seven 
radially arranged copies of hsp60. Our studies 
have demonstrated that unfolded mitochondrial 
proteins entering the matrix space become asso- 
ciated with the surface of the hsp60 complex. 
Then, in steps requiring both energy (ATP) and a 
second protein, the polypeptides are folded into 
their active forms and released from the 
complex. 
The folding pathway must be dictated by the 
amino acid sequence of the "substrate" protein 
to be folded, not by the hsp60 complex, because 
we have used the complex to fold proteins that 
normally reside outside mitochondria. It seems, 
however, that hsp60 acts by speeding up, or cata- 
lyzing, the folding of proteins. How does it do 
this? One possibility is that it simply prevents do- 
mains of proteins from wrongfully interacting, ei- 
ther with each other or with nearby proteins in 
the mitochondrial matrix, a "chaperone" func- 
tion. Another possibility is that the complex ac- 
tively promotes the progression of an unfolded 
protein through a series of folding steps. 
Because mitochondria arose from bacteria (one 
cell ingested another), it is not surprising that a 
structurally related component, the groEL pro- 
tein, has been found in bacteria. In the 1970s it 
was observed that when Escherichia coli cells 
partially defective in this protein are infected 
with a virus, the newly made viral coat proteins 
are unable to assemble to make new virus 
particles. 
Additional evidence comes from our reconsti- 
tution experiments. When a test protein unfolded 
in denaturant was diluted into a mixture contain- 
ing purified groEL complex, it became bound to 
the complex and was thus prevented from mis- 
folding and aggregating. The bound protein was 
found as a folding intermediate, called a molten 
globule, that has formed its local structures but 
lacks the three-dimensional organization of the 
active form. When both a cooperating compo- 
nent, groES (a small ring structure also with seven 
members), and ATP were added, the polypeptide 
was observed to reorganize its structure while in 
association with the complex during the next 
minutes and to be released in its active form. The 
cost of folding a single polypeptide was approxi- 
mately 100 ATP hydrolyzed, amounting to about 
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