Anfinsens Experiment

[sona224]

I was reading about Anfinsens experiment with the S and R bacteria strands and rats. I understand the results that came about when the S and R strands were injected separately and together.

In his supplementary experiment where he wanted to know if substances within cells cause proteins to fold to their 3-D shapes (the one where he used urea and mercaptoethanol), I don't understand why the results were different when he removed mercaptoethanol first vs. when he removed urea first.

If I understand everything correctly, when he removed mercaptoethanol first, there was no actvity and when he removed urea first there was activity. Why does it matter? Shouldn't it be the same? Or am I just not understanding the experiment?

Any help would be great!

[MrMistery]

can you please describe the experiment?
beta-mercaptoethanol is a reducing agent used to break disulfide bonds.

[sona224]

Anfinsen used the protein RNase A (has a single polypeptide chain with 8 cysteines). Anfinsen wanted to know if there are substances within cells (enzymes?) that cause other proteins to fold properly into their appropriate 3D shapes.

His first step was to apply mercaptoethanol alone to the RNase and that caused the S-S bonds between the cysteins to break, but no unfolding. Then he applied urea alone and that caused unfolding but didn't change the sequence of the amino acids. Both mercaptoethanol and urea caused the RNase to lose it's enzymatic function.

The following part I'm having trouble understanding. So he decided to remove these reagents (urea and mercaptoethanol) in 2 ways:
Method 1: remove mercaptoethanol first and then remove urea
Method 2: remove urea first and then remove mercaptoethanol

Method 1 resulted with very little activity in the RNase. Method 2 resulted with the RNase regaining almost all of it's enzymatic function.

What I'm having trouble understanding is, if in the end, both reagents are removed, then how come removing one reagent results in the protein regaining it's enzymatic function? Why does it matter what order the reagents are removed? In the end shouldn't it be the same?

:-/

[MrMistery]

ok, here's the issue. it's quite easy to grasp actually.
Urea is a denaturing agent, because it's amino groups interact with a protein's tertiary structure, thereby denaturing it.
What the guy did is this: he broke the S-S bonds with mercaptoethanol and denatured the protein with urea. I'm gonna strat with method two because it is easier to understand.

Method 2: removing the urea allows the protein to renature, returning to it's initial 3D conformation. Then, when the mercaptoehanol is removed, the S-S bonds form back, in the same way they were intially. This allows the protein to regain its function

Method 1: removing the mercaptoethanol causes the S-S to form back, but because the protein is still denatured by the urea, the bonds form in a different way. The cisteines connect to one another in a different way than they originally did. Then the urea is removed. Even though the denaturing agent is no longer present, the protein cannot return to its initial 3D structure because the S-S bonds are wrong. therefore function is lost.

[hisunny4u]

Anfinsen performed a series of experiments on ribonuclease A, a small protein with an activity that could be easily assayed

Instead of temperature, a strong solution of urea was used to denature the protein by destabilizing the structure of water, allowing it to solvate nonpolar groups

2-mercaptoethanol was also required, to reduce the 4 disulfide linkages of the native conformation

The experiment showed that if the disulfide bonds were allowed to form in the denatured state, this would trap the protein in a non-native and inactive conformation (how many possible alternatives?)

if u still have a doubt u can follow the attached file for animation


CONCLUSIONS: HE TRIED EXPERIMENTALLY TO SHOW THAT PROTEINS CAN BE RENATURED EVEN AFTER DENATURATION AND PROTEIN FOLDING INVOLVS MANY RANDOM CONFORMATIONS AND PROTEIN TRIES MANY CONFORMATIONS UNTIL A STABLE CONFORMATION IS FORMED ..........if u still have doubts u can mail me

[kikkid]

If heat was used as opposed to chemicals, would that completely destroy the protein, leaving no way for it to regenerate after the temperature was restored to normal? Are there other examples of this experiment, say ones that are a bit more modern?