3.

1
Bringing an Enzyme
Back to Life
B
y the 1950s, scientists realized that DNA held the code that allowed
proteins to be synthesized. Nevertheless, how a chain of amino
acids folds into a fully functional protein, with the proper three-
dimensional structure, remained a mystery. A mechanism must exist
to assure the proper folding of the protein. But where did that
information come from? In 1957, Christian Anfinsen published the first
evidence that the information for proper folding was held within the
protein itself.
Background
Proteins are made from combinations of 20 amino acids that
then fold into complex structures. The unfolded amino acid
chain is called the primary structure. To have biological ac-
tivity, the protein must fold into proper secondary and ter-
tiary structures. These structures are held together by chem-
ical interactions between the side chains of the amino acids,
including hydrogen bonds, hydrophobic interactions, and,
at times, covalent bonds. How these higher structures form
has long been a mystery. Does the protein fold correctly as
it is synthesized or does it require the action of other pro-
teins to correctly fold it? Can it correctly fold on its own
spontaneously?
In the 1950s, Anfinsen was a biochemist interested in
the proper folding of proteins. Specifically, he was investi-
gating the formation of disulfide bridges, which are cova-
lent bonds between cysteine side chains that serve as one of
the major anchors holding together the structure of secreted
proteins. He believed that the protein itself contained all the
information necessary for proper protein folding. He pro-
posed the thermodynamic hypothesis, which stated that
the biologically active structure of a protein was also the
most thermodynamically stable under in vivo conditions. In
other words, if the intracellular conditions could be mimic-
ked in a test tube, then a protein would naturally fold into
its active conformation. He began his work on a secreted
enzyme, bovine pancreatic ribonuclease, and studied its abil-
ity to properly fold outside of the cell.
The Experiment
Proteins perform a wide variety of functions in the cell. Re-
gardless of its function, a protein must be properly folded
to carry out its biological role. For protein folding studies
it is best to study an enzyme whose biological activity can
be easily monitored by performing in vitro. Anfinsen chose
a small, secreted protein, the enzyme ribonuclease, in which
he could monitor proper folding by assaying its ability to
catalyze the cleavage of RNA.
Ribonuclease, a secreted protein, is active under oxidiz-
ing conditions in vitro. The tertiary structure of active ri-
bonuclease is held together by four disulfide bridges. Adding
a reducing agent, which reduces the disulfide bond between
two cysteine side chains to two free sulfhydryl groups, can
disrupt this covalent interaction. Complete denaturation of
ribonuclease requires treatment with a reducing agent.
Anfinsen monitored the reduction of ribonuclease by mea-
suring the number of free sulfhydryl groups present in the
Classic Experiment
TABLE 3-1 Cell-free Refolding of Ribonuclease
Activity as a Percent of
Concentration of Protein Equivalent Concentration
(mg/ml) of Native Ribonuclease
7.0 31%
4.8 70%
2.3 75%
0.9 77%
0.35 94%
[Data adapted from C. B. Anfinsen and E. Haber, 1961, Journal of
Biological Chemistry 236:1362.]
protein. In the oxidized state, there are no free sulfhydryl
groups in ribonuclease because each cysteine residue is in-
volved in a disulfide bond. In the completely reduced state,
on the other hand, ribonuclease contains eight free
sulfhydryl groups. Anfinsen exploited this difference to as-
sess the extent of reduction by using spectrophotometric as-
say to titrate the number of sulfhydryl groups.
To study protein folding outside the cell, one must first
denature the protein. Proteins are easily denatured by heat,
mechanical disruption such as shaking, and chemical treat-
ment. Proteins with disulfide bridges require an additional
measure of treatment with a reducing agent to break apart
these covalent bonds. To denature ribonuclease, Anfinsen
first reduced the disulfide bridges with thioglycolic acid. He
then denatured the protein by using a high concentration of
urea and incubating the solution at room temperature. He
demonstrated that this treatment rendered the enzyme in-
active by showing that ribonuclease was now unable to cat-
alyze the cleavage of RNA. Using the spectrophotometric
assay, he went on to show that the inactive ribonuclease con-
tained eight sulfhydryl groups, which corresponded to the
four broken disulfide bridges. With a completely reduced,
denatured protein in hand, Anfinsen then could ask: Can a
denatured enzyme correctly fold in vitro and become active
again?
To find the answer, Anfinsen allowed a solution of re-
duced, denatured ribonuclease to oxidize. He removed the
urea from the denatured enzyme by precipitation. Next, he
resuspended the urea-free denatured ribonuclease in a
buffered solution and incubated it for two to three days. Ex-
posure to molecular oxygen in the atmosphere oxidized the
cysteine residues. He then compared the activity of this re-
natured ribonuclease to that of the native enzyme. In initial
experiments, 1219 percent of the previously inactive pro-
tein were able to catalyze the cleavage of RNA once again.
Proteins aggregate at high concentrations, which makes it
difficult for them to fold properly. By decreasing the over-
all concentration of ribonuclease in solution, Anfinsen
showed that up to 94 percent of the protein could be re-
folded (see Table 3.1). The enzyme had folded back to its
active conformation outside of the cell, demonstrating that
the information for the protein folding is contained in the
protein itself.
Discussion
Through careful experiments, Anfinsen demonstrated that
the information required to properly fold a protein is con-
tained in its primary sequence. His careful analysis of the
chemistry of this process answered a fundamental question
in biology. He went on to demonstrate the cell-free refold-
ing of other enzymes, including proteins lacking disulfide
bridges. While it is possible to properly fold a number of
proteins outside of the normal protein-processing machin-
ery in the cell, this process is greatly accelerated in vivo by
a number of enzymes. Anfinsen continued to study the
protein-folding problem. Although the thermodynamic
hypothesis does not hold true for all proteins, Anfinsens
demonstration of the cell-free refolding of ribonuclease made
a mark on the field of biochemistry. In 1972, he received
the Nobel Prize for Chemistry for his work.