Anfinsen Experiment - Video Tutorials & Practice Problems
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Anfinsen Experiment
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So now that we know about protein de nature ation, we can talk about a critical experiment to our understanding of protein structure, theano fence and experiment. So, way back in the 19 fifties, there was this guy named Christian and fencing that performed these experiments that demonstrated to major principles of protein structure, and the first major principle that he demonstrated was just 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 and Fenson actually demonstrated this with his experiments. Now, the second major principle that Christian infants and demonstrated was that a protein actually spontaneously folds into its native confirmation or its native, uh, state. And so what this means is that protein folding is an extra gone IQ process and a thermo, dynamically favorable process. And so, um, 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. And so in Christian and Vinson's experiment, he actually used the to re agents Yuria and Baltimore Capito, Ethanol and hey used them to affect the protein structure of a protein known as robo nucleus A and Revo nucleus. A is abbreviated with RNA say, And so, as we already know from our previous lessons, we know that the addition of both Yuria and bottom or capital ethanol is going to result in the d nature ation of the robo nucleus. A. And we also know that removal of both your re embodiment capital ethanol is going to re nature R N s A and so Christian. And Fenson actually demonstrated this and his experiments, and it really showed that it's really the primary sequence off Arnas A that dictates its proper folding. Now, what and Fenson also did was after he added, Yuria and bottom are kept to ethanol and denatured Arna say he subsequently removed on Lee the bottom or capital ethanol and he kept the Yuria in there and what ended up happening was he formed a scrambled protein and scrambled protein had random di sulfide bonds or random die sulfides. And what that showed is that it's really the non Covalin interactions that are required to form the proper die sulfide bonds and we'll be able to see that in our example down below as well. So in our example, down below, What we have is the and Fenson Revo nucleus, a experiment. And so up here the top left. What we have is our normal protein and so notice with our normal protein here that it has its specific structure that looks like this and which will see, is that there are proper die sulfides that are being formed So all of the dice sulfides are shown. So there are four die sulfide bonds in ribbon. Nucleus is structure and they're highlighted here. And so notice that the dye sulfides formed between the appropriate residue. So the way that we know that is that they are color coded. So the green forms with the green, the pink forms of the pink, the orange with the orange and the blue with the blue. And so that means that we have proper die sulfides and we have our proper structure. And that means our normal protein is gonna be catalytic Lee active. It's gonna be catalytic lee active, which means that it's going to be performing its job correctly, it's gonna be functioning properly. And so what happens is if we take, um are normal protein and we add both Yuria and bottom or Capito ethanol, which is this red arrow here. We know that your area is going to disrupt all the non Covalin bonds and bottom, or Capito. Ethanol is going to break all of our die sulfide bones, and so that ends up resulting in a denatured protein, which is what we have over here on the right. We have a denatured protein and notice that it does not have the same structure as it did over here and in our denatured protein because we disrupted and we broke all of these die sulfide bonds noticed that there are no dice sulfide bonds over here that we have also self hydro groups because our die sulfide bonds have been reduced. And so what you'll see is that our protein are denatured protein. Is catalytic lee inactive, which means that it does not function anymore. It does not work, it does not do its job anymore. But what and Fenson was able to do was he took this denatured protein and he removed the Yuri Um bottom a capital ethanol through a process known as dialysis, which is a technique that we'll talk about later on in our course. But just know that he removed the Yuria and body armor capital ethanol. And of course, what that resulted in was re natural ation off the protein. So it was able to reform its exact structure. And so it was able to regain its catalytic activity so that it was ableto work, and that showed that the only thing that was required for the protein to fold was its primary structure. It retain its primary structure, and it was able to re fold. And another thing that it showed was that the protein, even though it could have folded into a completely different fold, it folded into its most stable fold, which is its native fold. So it showed that the native fold, or the native confirmation, is the most thermodynamic lee favorable confirmation. And it also showed that, uh, protein folding is spontaneous and ex organic. And so what? And Fenson was also able to show, was if he took his denatured protein, which had Yuria and bottom or capital ethanol in it. And if he just Onley removed the bottom or capital ethanol. So if he Onley removed bottom or capital ethanol and he kept the Yuria So the Yuria stays. What happens is the non Covalin bonds are still not gonna be able to form. But by removing the bottom or capito ethanol, the dye sulfide bonds are able to reform. But what he noticed was that this actually resulted in a scrambled protein and that the dye sulfide bonds did not form in the proper location. So what happened was there were random die sulfides and we could see that in our image because noticed that with our image that the dye sulfides are not matching up, we don't have the green bonding with the green. We have green bonding with pink and we have orange bonding with blue blue bonding with green. They're not properly matched up. So these guys sulfides, they did reform, but they reformed randomly and what that showed was that really it's the non Covalin interactions that air required for the dice. Sulfide bonds toe form properly. And so this protein down here is actually catalytic Lee inactive, which means that it does not work and it might have a little bit more activity than the denatured protein up here. It might have a little bit more catalytic activity. Maybe it has 1% activity instead of 0% but it's still, for the most part, Catalytic Lee inactive. And it doesn't work for the most part. And that shows that we really need all of our co violent and non covalin bonds to be working for us to get catalytic activity. So then what and Fenson did was he, um, removed all of the Yuria that was present in the scramble protein. And then he added, back in just a small amount, he added a tiny amount of Baltimore capital ethanol back in just a little bit so that all of these bonds would break. But they could still reform. And over a long period of time, what ended up happening was it re natured the protein back to its original form. And so this showed that again, the it's the non covalin interactions that allow for the dye sulfide bonds to form in the proper location. So now the dice sulfides air back into their proper locations, and so really, this is the and fence and experiment, and the major thing that you want to take away is that the infants and experiment demonstrated that primary protein structure dictates the tertiary protein structure and that a protein will always fold into its native confirmation, which is its most stable state in it will fold spontaneously into that state. And so this concludes our lesson on the infants and experiment, and we'll be able to get a bunch of practice and our practice video so I'll see you guys there.
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Problem
Problem
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.
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Problem
Problem
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.
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Problem
Problem
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.
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Problem
Problem
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.