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C L A S S I C E X P E R I M E N T 3 . 1

BRINGING AN ENZYME BACK TO LIFEC. B. Anfinsen and E. Haber, 1961, Journal of Biological Chemistry236:1362

By the 1950s, scientists realized thatDNA held the code that allowed pro-teins to be synthesized. Nevertheless,how a chain of amino acids folds intoa fully functional protein, with theproper three-dimensional structure, re-mained a mystery. A mechanism mustexist to assure the proper folding of theprotein. But where did that informa-tion come from? In 1957, ChristianAnfinsen published the first evidencethat the information for proper foldingwas held within the protein itself.

Background

Proteins are made from combinationsof 20 amino acids that then fold intocomplex structures. The unfoldedamino acid chain is called the primarystructure. To have biological activity,the protein must fold into proper sec-ondary and tertiary structures. Thesestructures are held together by interac-tions between the side chains andbackbone atoms of the amino acids,including hydrogen bonds, hydropho-bic interactions, and, at times, covalentbonds. How these higher structuresform had long been a mystery. Doesthe protein fold correctly as it is syn-thesized or does it require the actionof other proteins to correctly fold it?Can it correctly fold on its own spon-taneously?

In the 1950s, Anfinsen was a bio-chemist interested in the proper fold-ing of proteins. Specifically, he wasinvestigating the formation of disulfidebridges, which are covalent bonds be-tween cysteine side chains that serveas one of the major anchors holdingtogether the structure of secreted pro-teins. He believed that the protein itselfcontained all the information neces-sary for proper protein folding. Heproposed the thermodynamic hypoth-esis, which stated that the biologicallyactive structure of a protein was also

the most thermodynamically stable un-der in vivo conditions. In other words,if the intracellular conditions could bemimicked in a test tube (in vitro), thena protein would naturally fold into itsactive conformation. He began his workon a secreted enzyme, bovine pancreaticribonuclease, and studied its ability toproperly fold outside of the cell.

The Experiment

Proteins perform a wide variety of func-tions in the cell. Regardless of its func-tion, a protein must be properly foldedto carry out its biological role. For pro-tein folding studies it is best to study anenzyme whose biological activity can beeasily monitored by performing a test,or assay of its activity in vitro. Anfin-sen chose a small, secreted protein, theenzyme ribonuclease, in which he couldmonitor proper folding by assaying itsability to catalyze the cleavage of RNA.

Ribonuclease, a secreted protein, isactive under oxidizing conditions invitro. The tertiary structure of active ri-bonuclease is held together by fourdisulfide bonds or bridges. Adding a re-ducing agent reduces the disulfide bondbetween two cysteine side chains to twofree sulfhydryl groups, and can disruptthis covalent interaction. Completedenaturation of ribonuclease requirestreatment with a reducing agent. An-finsen monitored the reduction ofribonuclease by measuring the numberof free sulfhydryl groups present in theprotein. In the oxidized state, there areno free sulfhydryl groups in ribonucle-ase because each cysteine residue isinvolved in a disulfide bond. In thecompletely reduced state, on the otherhand, ribonuclease contains eight freesulfhydryl groups. Anfinsen exploitedthis difference to assess the extent of re-duction by using a spectrophotometricassay to titrate, or count, the number offree sulfhydryl groups.

To study protein folding outsithe cell, one must first denature thprotein. Proteins are easily denatureby heat, mechanical disruption such shaking, and chemical treatment. Prteins with disulfide bridges require aadditional measure of treatment withreducing agent to break apart thecovalent bonds. To denature ribonclease, Anfinsen first reduced the disufide bridges with thioglycolic acid. Hthen denatured the protein by usinghigh concentration of urea and incubaing the solution at room temperaturHe demonstrated that this treatmerendered the enzyme inactive by showing that ribonuclease was now unabto catalyze the cleavage of RNA. Usinthe spectrophotometric assay, he weon to show that the inactive ribonuclase contained eight sulfhydryl groupwhich corresponded to the four brken disulfide bridges. With a completely reduced, denatured protein hand, Anfinsen then could ask: Candenatured enzyme correctly fold vitro and become active again?

To find the answer, Anfinsen alowed a solution of reduced, denatureribonuclease to oxidize. He removethe urea from the denatured enzyme bprecipitation. Next, he resuspendethe urea-free denatured ribonuclease a buffered solution and incubated for two to three days. Exposure molecular oxygen in the atmospheoxidized the cysteine residues. He thecompared the activity of this renaturribonuclease to that of the native ezyme. In initial experiments, 121percent of the previously inactive prtein were able to catalyze the cleavaof RNA once again. Proteins aggregaat high concentrations, which makesdifficult for them to fold properly. Bdecreasing the overall concentratioof ribonuclease in solution, Anfinseshowed that up to 94 percent of thprotein could be refolded (see Table 1

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The enzyme had folded back to its ac-tive conformation outside of the cell,

demonstrating that the information forthe protein folding is contained in theprotein itself.

Discussion

Through careful experiments, Anfin-sen demonstrated that the informationrequired to properly fold a protein is

contained in its primary sequence. Hiscareful analysis of the chemistry of thisprocess answered a fundamental ques-tion in biology. He went on to demon-strate the cell-free refolding of otherenzymes, including proteins lackingdisulfide bridges. While it is possible toproperly fold a number of proteins out-side of the normal protein-processingmachinery in the cell, this process isgreatly accelerated in vivo by a numberof proteins. Anfinsen continued tostudy the protein-folding problem. Al-though the thermodynamic hypothe-sis does not hold true for all proteins,Anfinsens demonstration of the cell-free refolding of ribonuclease made amark on the field of biochemistry. In1972, he received the Nobel Prize forChemistry for his work.

TABLE 1 Cell-free Refolding of Ribonuclease

ACTIVITY AS A PERCENT OF EQUIVALENT

CONCENTRATION OF PROTEIN (MG/ML) CONCENTRATION 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.]