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E X P E R I M E N T
3 . 1
BRINGING AN ENZYME BACK TO LIFE
C. B. Anfinsen and E. Haber, 1961, Journal of Biological Chemistry 236:1362
By 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.
the most thermodynamically stable under in vivo conditions. In other words, if the intracellular conditions could be mimicked in a test tube (in vitro), then a protein would naturally fold into its active conformation. He began his work on a secreted enzyme, bovine pancreatic ribonuclease, and studied its ability to properly fold outside of the cell.
Proteins perform a wide variety of functions in the cell. Regardless 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 a test, or “assay” of its activity 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 oxidizing conditions in vitro. The tertiary structure of active ribonuclease is held together by four disulfide bonds or bridges. Adding a reducing agent reduces the disulfide bond between two cysteine side chains to two free sulfhydryl groups, and can disrupt this covalent interaction. Complete denaturation of ribonuclease requires treatment with a reducing agent. Anfinsen monitored the reduction of ribonuclease by measuring the number of free sulfhydryl groups present in the protein. In the oxidized state, there are no free sulfhydryl groups in ribonuclease because each cysteine residue is involved in a disulfide bond. In the completely reduced state, on the other hand, ribonuclease contains eight free sulfhydryl groups. Anfinsen exploited this difference to assess the extent of reduction by using a spectrophotometric assay to titrate, or count, the number of free sulfhydryl groups.
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 activity, the protein must fold into proper secondary and tertiary structures. These structures are held together by interactions between the side chains and backbone atoms of the amino acids, including hydrogen bonds, hydrophobic interactions, and, at times, covalent bonds. How these higher structures form had long been a mystery. Does the protein fold correctly as it is synthesized or does it require the action of other proteins 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 investigating the formation of disulfide bridges, which are covalent 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 proposed the “thermodynamic hypothesis,” which stated that the biologically active structure of a protein was also
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 treatment. 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 inactive by showing that ribonuclease was now unable to catalyze the cleavage of RNA. Using the spectrophotometric assay, he went on to show that the inactive ribonuclease contained 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 reduced, 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. Exposure to molecular oxygen in the atmosphere oxidized the cysteine residues. He then compared the activity of this renatured ribonuclease to that of the native enzyme. In initial experiments, 12–19 percent of the previously inactive protein 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 overall concentration of ribonuclease in solution, Anfinsen showed that up to 94 percent of the protein could be refolded (see Table 1).
Haber. Although the “thermodynamic hypothesis” does not hold true for all proteins. Anfinsen demonstrated that the information required to properly fold a protein is contained in its primary sequence. Anfinsen and E. including proteins lacking disulfide bridges.9 0. In 1972.8 2.TABLE 1 Cell-free Refolding of Ribonuclease CONCENTRATION OF PROTEIN (MG/ML) 7. he received the Nobel Prize for Chemistry for his work. this process is greatly accelerated in vivo by a number of proteins.35 ACTIVITY AS A PERCENT OF EQUIVALENT CONCENTRATION OF NATIVE RIBONUCLEASE 31% 70% 75% 77% 94% [Data adapted from C. He went on to demonstrate the cell-free refolding of other enzymes. While it is possible to properly fold a number of proteins outside of the normal protein-processing machinery in the cell. B. . demonstrating that the information for the protein folding is contained in the protein itself. Anfinsen’s demonstration of the cellfree refolding of ribonuclease made a mark on the field of biochemistry.] The enzyme had folded back to its active conformation outside of the cell. His careful analysis of the chemistry of this process answered a fundamental question in biology.3 0. Journal of Biological Chemistry 236:1362.0 4. Discussion Through careful experiments. Anfinsen continued to study the protein-folding problem. 1961.