in this video, we're gonna talk about diagonal electrophoresis. So diagonal electrophoresis is another type of electro for Reese's technique, which means that it uses an electric field to separate proteins. Now, more specifically, diagonal electrophoresis is used by biochemist to isolate and identify die sulfide linked proteins, and they use it to determine the positions of original die sulfide bonds. And so recall from our previous lessons that die sulfide bonds are co Vaillant bonds that link the are groups of any to Sistine Residues. And these two Sistine residues could be present either on the same poly peptide chain and form a die sulfide bond. Or they could be present on separate poly peptide chains and co violently linked those separate poly peptide chains via di sulfide bomb and so down below. In our example of diagonal electrophoresis, we're gonna talk about how diagonal electrophoresis works. But just to give you guys a heads up on what to expect for the results of diagonal electrophoresis, it turns out that the peptides that do not have die sulfide bonds or the peptides without die sulfide bonds are actually going to align diagonally or a line on a diagonal line due to unchanged mobility. And so again, this doesn't make a lot of sense right now, but it will once we get to our example. And so what I want you guys to know is that you're gonna be looking for some kind of diagonal line and any proteins that lie on that diagonal line that a line on the diagonal line are going to not have any dye sulfide bond. There's air gonna be the peptides without die sulfide bonds. And so what that means is that peptides that do have die sulfide bonds so peptides with di sulfide bonds are actually going to lie off of the diagonal. So that means that they will not be on this diagonal line, and that is because they're going to have changed mobility. And so let's take a look at our example down below of diagnose electrophoresis to clear up this idea. And so notice that on the far left up here, what we have is our native protein and notice that our native protein has this very particular shape to it. It has its native protein structure, and we know that it's gonna have an end terminal end and it will also have a C terminal end on the opposite end. And so we know that when we're counting amino acids that we count them and we consider amino acids from the internal end to the C terminal in and so we can count amino acids and give them a particular position. So notice that this particular native protein here has a total of 3 16 residues, and they're located at position number 36 position number 54 position number 72. And so we can clearly see from this image that it's Sistine 36 16 54 that form a die sulfide bond, which is indicated here, and 16 72 does not form a die sulfide bomb. But let's say for just for the sake of this example, that the biochemist performing this experiment does not know which of these three Sistine are die sulfide bonded. And that's why the biochemist would perform diagonal electrophoresis to figure out which of these three sis teens are die sulfide bonded if any of them are, and so the very first step in diagonal electrophoresis is going to be to fragment the protein or cleave the protein and two fragments. And so the biochemist can do that by treating the native protein with either an enzyme such as a pep today's, which breaks down proteins. Or the biochemist could use some kind of chemical that will cleave the protein down into fragments. And so, of course, after we cleave the protein into fragments, that's going to result in a bunch of protein fragments. And so notice that we have a protein fragment over here that has a 72 which again we know does not have a dice sulfide bond, and then we have another fragment over here. But notice that this fragment has a di sulfide bond between 36 and 16 54. And so these two fragments, even though their backbones air separated, they're still co violently linked via the dice sulfide bond. And so, after we cleave are protein into fragments. The second step is going to be two separate all of these protein fragments via STS page, and we know the STS page separates proteins based on their molecular size, and so notice that all of these protein fragments have different molecular sizes. So STS Page is able to separate all of these proteins based on their molecular size. Now, notice that the protein that contains the dye sulfide bond here is going to migrate together. So these two peptide fragments are gonna migrate together because they're die sulfide length. And that means that they're gonna show up as a single band in the S. D s page gel, even though there are two fragments. And so that's important to keep in mind as we move along through our process. So after we separate our protein fragments, uh, in STS page, the third step, which is down below here, is to expose all of the proteins that air inside of our gel over here to perform IC acid vapors and the performing acid vapors will. Def you They're literally a gas that will diffuse into the gel and interact with the proteins. And they when they interact with the proteins, they're going to cleave all of the die sulfide bonds. And so essentially, you can think of performing acid as having the same or a similar effect as bottom or Capito ethanol, which also cleaves die sulfide bonds and down below. What we have is an image of the performing acid structure so we can label it as performing acid. And so, after we expose our gel and the protein fragments to perform it acid, what's gonna happen is all of the dice sulfide bonds are gonna be cleaved. Which means that our fragment up here, which had a die sulfide bond, is no longer going to have a die sulfide bond linking those two chained together. And so, in the fourth step, all we need to do is take our STS page, yell at the top up here, and we can take it and literally just turn it sideways. And when we turn it sideways, weaken, run STs page a second time. But this time we run STS page and the perpendicular direction. And so what you'll see is that when we run STS page in the perpendicular direction, we finally see that diagonal line that we knew that we were gonna see earlier. We were expecting to see a diagonal line, and there it is. This is our diagonal line. And so recall up above we said that with the diagonal line that we see, it's the peptides that do not have die sulfide bonds that align diagonally so all of the peptide fragments that are in this diagonal, all of these do not have die sulfide bonds. But the ones that lie off of the diagonal line do have die sulfide bonds. That would be these two fragments here. And so the reason that all of these peptides aligned diagonally is because their mobility from the first STS page to the second STS page down below, is unchanged. Nothing really changed for those peptides from the first gel to the second gel, and so they run the same distance, and they all form a diagonal line. However, the peptides that lie off of the diagonal line they're migrating differently from the first STS page to the second STS page. And that's why they lie off of the diagonal. And the reason that they run differently from the first to the second STS is because of this third step that's in between where we added performing acid, which cleaved the dye sulfide bonds. So now that they don't have that die sulfide bond linking them, they will run according to their actual sizes. So you've got the smaller fragment that will move faster, and another fragment here that's also gonna move faster, and so that's why they lie off the line. So an important take away is that it's the protein bands that lie off of the diagonal line that are actually die sulfide length. So it's thes proteins that the biochemist is gonna be interested in if they're trying to determine the position of the diesel five bonds. And so it makes it pretty easy to see visually that these are the ones that are interested in because all of the other ones lie on this diagonal so we can take these to die sulfide link proteins, and we can move them into our fifth and final step. And the fifth and final step is toe. Take these die sulfide link peptides, isolate them from the gel and then subject them to sequencing. And the sequencing technique is what really reveals the dice sulfide positions. But it would have been it's the diagonal electrophoresis that allowed us to obtain the dice sulfide linked peptides in the first place and so up above. What we can say is that the very fifth and final step is that peptides that are found to be die sulfide linked can be isolated, sequenced to determine the dye sulfide bond positions. And so you can see here how diagonal electrophoresis can be a really useful tool toe. Help biochemist narrow down which fragments actually have a die sulfide bond in which fragments do not. And so, after sequencing those two fragments, the biochemist would be able to reveal that it's 16 36 and 54 that form a die sulfide bond, and 16 72 would not form a die sulfide bond. And so that concludes our lesson on diagonal electrophoresis, and we'll be able to get some practice in our next video, so I'll see you guys there.
Which of the following techniques is used specifically to help determine the site of a disulfide bond?
MALDI-TOF Mass spectrometry.
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So in our last lesson video, we said that diagonal electrophoresis can isolate peptide fragments that are die sulfide linked, and it can be used to help determine the original position of die sulfide bonds. Now, in this video, we're going to talk about how diagonal electrophoresis can distinguish between in ter Ching dai sulfide bonds as well as intra chain die sulfide bonds. And so it turns out that diagonal electrophoresis results actually differs a little bit for interchange I sulfide bonds, which are die sulfide bonds that form between 16 residues found on separate poly peptide chains, as well as intra chain die sulfide bonds which are die sulfide bonds that form between Sistine Residues found on the same poly peptide chain. And so it turns out that upon cleavage of Onley, the interchange I sulfides using performing acid leads to the peptide fragments, getting smaller and ultimately traveling faster in the gel and so upon cleavage of Onley, the entra chain die sulfides using performing acid. The peptide fragments will actually change their shape and travel slower in the gel. And so let's take a look at our example down below of the interchange versus the interchange I sulfides in diagonal electrophoresis to clear up this idea. And so we know that the very first step in diagonal electrophoresis is to cleave our native protein down into a bunch of different fragments. And so when we cleave, are peptide into a bunch of fragments, all of the die sulfide bonds remain intact and nothing happens to them. And so the second step, we run our protein fragments using STS page, and we separate the protein fragments based on their size. And so, uh, that STS page keeps all of the die sulfide bonds intact. And so what you can see is we have a bunch of different protein fragments that are being shown here, and they go from p one all the way through peace seven and which will notice is that all of the dice sulfide bonds are remaining intact for the first gel electrophoresis of diagonal electrophoresis. And so, which will notice is the first to die sulfide bonds that are present. We are color coding and yellow because they are interchange I sulfides that formed between separate poly peptide chains. And so you can see that p one is forming a die sulfide bomb with P two and P four is forming a die sulfide bond with P five and those are interchange I sulfides whereas with P six here protein fragments. Six. We have an intra chained I sulfide because it's forming within the same poly peptide chain. Now, after the first direction of electrophoresis, we know that the third step of diagonal electrophoresis is to expose all of these peptide fragments to perform ic acid and perform it acid cleaves all of the dice sulfide bond so we can see that uh, what we have on this axis is the second direction of electrophoresis. After the dye sulfide bonds have been cleaved and so which will notice is inside of our gel. There are no mawr die sulfide bonds that air found. And so when we run our second direction of electrophoresis, we know that the peptides fragments that do not have any dye sulfide bonds such as peptide fragment three and peptide fragments seven are going to align diagonally on a diagonal line. And that's exactly what we see in our diagonal electrophoresis gel that peptide fragment three and peptide fragments seven, which are the Onley to that do not have any dye sulfides. They align diagonally on this line and any of the peptide fragments that do not align on the fragment, such as all of these other peptide fragments that are listed here. They do contain some kind of die sulfide bomb. So now, to distinguish between the inter chain and the interchange die sulfide bonds, so notice that what we said previously is that the peptide fragments that contain interchange I sulfide such as this one here and this one over here those peptide fragments will get smaller and ultimately travel faster in the gel. And because they travel faster in the gel, notice that they are below the diagonal line. These fragments because they contained, uh, die sulfide bonds uh, interchange I sulfide bonds. They became smaller and traveled faster. And that's why we find them below the, uh, diagonal line. Now, within tra Changi sulfide bonds such as the one with peptide fragments. Six. Notice that it results in, uh, changing the shape of the peptide fragment, and that leads to the peptide fragment traveling slower. And that's what we exactly what we see down below that when the intro Ching dai sulfide bond is cleaved peptide fragments. Six changes its shape and it changes its shape in such a way that it travels slower through the gel than it did the first time through electrophoresis. And so that's why it shows up above the diagonal line it shows up above. So essentially, what we're saying is that the way that we distinguish between inter chain and intra ching dai sulfides and diagonal electrophoresis is by looking at the migration of the peptides relative to the diagonal line that forms and so peptides that migrate faster, which are these peptides here are going to have some kind of interchange. I sulfide bond and peptide fragments that migrate slower are going to have an intra chain die sulfide. And so this concludes our lesson here on the difference between inter chain and interchange I sulfides and diagonal electrophoresis results, and we'll be able to get a little bit of practice in our next video. So I'll see you guys there
In the plot below, circle the point(s) representing peptides with intrachain disulfides.
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A gene encoding a protein with a single disulfide bond undergoes a mutation that changes a serine residue into a cysteine residue. You want to find out whether the disulfide pairing in this mutant is the same as in the original protein. Briefly layout a diagonal electrophoresis experiment to determine the answer.