The Anfinsen experiment, conducted by Christian Anfinsen in the 1950s, significantly advanced our understanding of protein structure, particularly the relationship between primary and tertiary structures. Anfinsen demonstrated two key principles: first, that the primary structure of a protein dictates its tertiary structure, and second, that proteins spontaneously fold into their native conformation, which is the most thermodynamically stable state.
In his experiments, Anfinsen used ribonuclease A (RNase A) and two reagents, urea and beta-mercaptoethanol. Urea disrupts non-covalent interactions, while beta-mercaptoethanol breaks disulfide bonds. The addition of both agents leads to the denaturation of RNase A, resulting in a loss of its specific structure and catalytic activity. This denatured protein contains only sulfhydryl groups, as the disulfide bonds have been reduced.
Upon removing both urea and beta-mercaptoethanol through dialysis, Anfinsen observed that RNase A could renature, regaining its original structure and catalytic function. This finding underscored that the primary sequence alone is sufficient for proper folding, as the protein returned to its native state, which is the most stable and energetically favorable conformation.
In a further experiment, Anfinsen removed only beta-mercaptoethanol while keeping urea present. This resulted in a scrambled protein with incorrectly formed disulfide bonds, indicating that non-covalent interactions are essential for the correct formation of disulfide linkages. The scrambled protein exhibited minimal catalytic activity, demonstrating that both covalent and non-covalent interactions are crucial for functional proteins.
Ultimately, Anfinsen's work illustrated that proteins inherently fold into their native conformations spontaneously and that the correct formation of disulfide bonds relies on the presence of non-covalent interactions. This experiment laid the groundwork for understanding protein folding and stability, emphasizing the importance of primary structure in determining the overall protein architecture.
