Anfinsen Experiment - Online Tutor, Practice Problems & Exam Prep

The Anfinsen experiment demonstrated that primary protein structure dictates tertiary structure, showing that proteins spontaneously fold into their native conformation, which is the most thermodynamically stable state. Using ribonuclease A, Anfinsen illustrated that denaturation occurs with urea and beta mercaptoethanol, disrupting non-covalent and disulfide bonds. Upon removal of these reagents, the protein renatured, regaining catalytic activity, emphasizing the importance of both covalent and non-covalent interactions in proper protein folding.

Anfinsen Experiment

So now that we know about protein denaturation, we can talk about a critical experiment to our understanding of protein structure, the Anne Fenson experiment. So, way back in the 1950s, there was this guy named Christian Anfinson who performed these experiments that demonstrated two major principles of protein structure. The first major principle that he demonstrated was that primary protein structure dictates the tertiary structure and we already knew that, right? We know that primary protein structure dictates all the other levels of protein structure. And so, Christian Anfinsen actually demonstrated this with his experiments. Now, the second major principle that Christian Anfinsen demonstrated was that a protein actually spontaneously folds into its native conformation or its native state. And so, this means that protein folding is an exergonic process and a thermodynamically favorable process. It also shows that when a protein goes to fold, its native state or its native fold is actually going to be its most stable state or the state that has the lowest amount of energy.

In Christian Anfinsen's experiment, he used two reagents, urea and beta mercaptoethanol, and he used them to affect the protein structure of a protein known as ribonuclease A, abbreviated as RNase A. As we already know from our previous lessons, the addition of both urea and beta mercaptoethanol results in the denaturation of ribonuclease A, and the removal of both reagents renatures RNase A. Christian Anfinsen demonstrated this in his experiments, showing that it is really the primary sequence of RNase A that dictates its proper folding.

What Anfinsen also did was, after he added urea and beta mercaptoethanol and denatured RNase A, he subsequently removed only the beta mercaptoethanol and kept the urea. What ended up happening was he formed a scrambled protein with random disulfide bonds. This showed that it is really the non-covalent interactions that are required to form the proper disulfide bonds. We'll be able to see that in our example down below as well.

In our example down below, we have the Anfinsen Ribonuclease A Experiment. At the top left, our normal protein has its specific structure with the proper disulfides formed between the appropriate residues. These disulfides form between the residues that are color coded: the green with the green, the pink with the pink, the orange with the orange, and the blue with the blue. This means we have proper disulfides and a proper structure, making our normal protein catalytically active and functioning properly.

When we add both urea and beta mercaptoethanol (shown by the red arrow), urea disrupts all the non-covalent bonds, and beta mercaptoethanol breaks all of the disulfide bonds, resulting in a denatured protein. In this state, there are no disulfide bonds; all we have are sulfhydryl groups since our disulfide bonds have been reduced. Our protein, now denatured, is catalytically inactive and does not function.

However, Anfinsen was able to renature the protein by removing the urea and beta mercaptoethanol through dialysis, allowing it to refold into its exact structure and regain catalytic activity. This demonstrated that the only thing required for the protein to fold was its primary structure. It also showed that, even though the protein could have folded into a completely different fold, it folded into its most stable fold, which is its native fold, indicating that the native conformation is the most thermodynamically favorable conformation and that protein folding is spontaneous and exergonic.

If Anfinsen took his denatured protein (with urea and beta mercaptoethanol) and removed only the beta mercaptoethanol, keeping the urea, disulfide bonds were able to reform by removing beta mercaptoethanol. However, these disulfide bonds reformed randomly, resulting in a scrambled protein. The disulfides did not match up properly, and this scrambled protein was mostly catalytically inactive, showing again that both covalent and non-covalent interactions are necessary for proper catalytic activity.

Finally, Anfinsen removed all the urea from the scrambled protein, added back a small amount of beta mercaptoethanol, and allowed the bonds to break and reform over time. This process renatured the protein back to its original form, confirming that non-covalent interactions are crucial for disulfide bonds to form in the proper location.

In summary, the Anfinsen experiment demonstrated that primary protein structure dictates the tertiary protein structure, and that a protein will always fold into its native conformation, which is its most stable state. This concludes our lesson on the Anfinsen experiment, and we'll be able to get a bunch of practice in our practice video. So I'll see you guys there.

Which of the following conclusions could Anfinsen draw from his RNase A experiment?

• A. Disulfide bridges are unnecessary for the function of RNase A.
• B. Kinetics is the main barrier to a protein adopting its native fold.
• C. Proteins spontaneously adopt their native fold, which specifies location of disulfide bridges.
• D. RNase activity cannot be destroyed by urea alone at any concentration.

What is likely to happen to Ribonuclease A if it is treated with both urea & β-mercaptoethanol?

• A. RNase A will denature and oxidize its disulfides to generate sulfhydryl groups.
• B. RNase A renatures but disulfide bonds are formed randomly between Cys residues.
• C. RNase A will denature and reduce its disulfides to generate sulfhydryl groups.
• D. RNase A will denature and oxidize its sulfhydryl groups to generate disulfides.

Which of the following occurred when RNase A properly refolded from a denatured state?

• A. The primary structure of the protein was rearranged.
• B. Most of the charged, hydrophilic residues were found buried in the core of the protein.
• C. The entropy of the protein structure itself was significantly increased.
• D. None of the above.

Which statement best supports the theory that primary protein structure dictates folding into its native state?

• A. RNase A loses all enzymatic activity upon denaturing in 8M urea.
• B. RNase A regains enzymatic activity upon removing urea & β-ME.
• C. Purified RNase A has 100% enzymatic activity in vitro.
• D. A reducing agent such as β-ME destroys disulfide bonds & eliminates RNase A enzymatic activity.

Here’s what students ask on this topic:

What is the Anfinsen experiment and why is it important?

The Anfinsen experiment, conducted by Christian Anfinsen in the 1950s, demonstrated that the primary structure of a protein dictates its tertiary structure. Using ribonuclease A, Anfinsen showed that proteins can spontaneously fold into their native conformation, which is the most thermodynamically stable state. He used urea and beta mercaptoethanol to denature the protein, disrupting non-covalent and disulfide bonds. Upon removal of these reagents, the protein renatured and regained its catalytic activity. This experiment highlighted the importance of both covalent and non-covalent interactions in proper protein folding, fundamentally advancing our understanding of protein chemistry.

How did Anfinsen demonstrate that primary structure dictates tertiary structure?

Anfinsen demonstrated that primary structure dictates tertiary structure by using ribonuclease A (RNase A). He denatured the protein with urea and beta mercaptoethanol, which disrupted non-covalent and disulfide bonds. When these reagents were removed, the protein spontaneously refolded into its native, catalytically active form. This showed that the primary amino acid sequence contains all the information necessary for the protein to fold into its correct three-dimensional structure, emphasizing the role of primary structure in determining tertiary structure.

What reagents did Anfinsen use in his experiment and what was their purpose?

Anfinsen used urea and beta mercaptoethanol in his experiment. Urea was used to disrupt non-covalent interactions, while beta mercaptoethanol was used to break disulfide bonds. These reagents denatured ribonuclease A (RNase A), rendering it catalytically inactive. By subsequently removing these reagents, Anfinsen was able to show that the protein could renature and regain its catalytic activity, demonstrating that the primary structure of the protein dictates its proper folding and function.

What did Anfinsen's experiment reveal about protein folding and stability?

Anfinsen's experiment revealed that protein folding is a spontaneous and exergonic process, meaning it occurs naturally and releases energy. It also showed that proteins fold into their native conformation, which is the most thermodynamically stable state. This native state is achieved through the proper formation of both covalent (disulfide bonds) and non-covalent interactions. The experiment underscored that the primary amino acid sequence of a protein contains all the information necessary for it to fold correctly and function properly.

Why did Anfinsen use ribonuclease A in his experiment?

Anfinsen used ribonuclease A (RNase A) in his experiment because it is a well-characterized enzyme with a known structure and catalytic activity. RNase A has four disulfide bonds, making it an ideal candidate to study the effects of denaturation and renaturation. By using RNase A, Anfinsen could clearly demonstrate the role of primary structure in dictating the protein's tertiary structure and its ability to regain catalytic activity after denaturation, thus providing strong evidence for his conclusions.