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Anfinsen's Experiment of Protein Folding
In the 1950s, Christian Anfinsen conducted a series of experiments in which he determined that all the information needed to form the three-dimensional structure of the polypeptide is stored in the specific sequence of amino acids in that polypeptide. Later experiments confirmed this fact - that primary structure determines the final conformation of the protein. Christian's plan was to use the appropriate denaturing agents, namely urea and beta mercaptoethanol, to break down the secondary and tertiary structure of ribonuclease, a polypeptide that consists of 124 amino acids and which contains four disulfide bonds within its tertiary structure. The urea agent is used to break down non-covalent bonds such as hydrogen bonds holding the secondary structure while the beta-mercaptoethanol was used to reduce and break down the disulfide bonds holding the tertiary structure together. In his first experiment, Christian exposed the native enzyme to excess beta mercaptoethanol and 8.0 M urea and he found that the protein was completely denatured. When he removed the two agents simultaneously via dialysis, he found that the protein refolded back into its original biologically active form. In his second experiment, instead of removing the two agents at the same time, he first removed the beta mercaptoethanol first and then removed the urea. What he discovered was that the final protein refolded but became scrambled and was no longer biologically active. This happened because the non-covalent bonds could not form in the presence of urea and so the disulfide bonds formed incorrectly (the non-covalent bonds coded by the primary sequence of amino acids are needed to direct the correct formation of the disulfide bonds). In his third experiment, he found that if he exposed the scrambled, inactive protein to trace amounts of beta mercaptoethanol in the absence of urea, the biologically active native structure eventually reformed. Why did this happen? Well the tiny amount of beta mercaptoethanol was enough to catalyze the breaking of the incorrect disulfide bonds. Eventually the protein formed the correct disulfide bridges and returned to its native form because this was thermodynamically most stable and lowest in energy form.
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