A gene encoding a protein with a single disulfide bond undergo...

Question

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.

Accepted Answer

So this practice problem says, a gene encoding a protein with a single disulfide bond undergoes a mutation that changes a serine residue into a cysteine residue, and we know that cysteine residues are capable of forming disulfide bonds. And so you wanna find out whether or not the disulfide pairing in this mutant is the same as in the original protein. And it says to briefly lay out a diagonal electrophoresis experiment to determine the answer. And so notice down below, I've provided a little image of our protein. So on the left, we have the original protein and on the right, we have the mutant protein. And we know that the original protein only has a single disulfide bond, and so it's shown in red right here at this position. And what we also know is that our original protein has a serine residue. And so notice that we've shown the serine residue over here in blue. Now with the mutant protein on the right, we know that the serine residue was on the original protein was mutated into a cysteine residue as it says up above. And so notice we have an additional cysteine shown in green. And so we wanna find out whether the disulfide pairing in the mutant is the same as in the original. So we wanna know, are these same 2 cysteines cysteines forming a disulfide like the original, or are there different cysteines forming a disulfide? And so, we know that diagonal electrophoresis is capable of isolating peptide fragments that are disulfide linked, and we can use it to help us determine the position of a disulfide bond. So we want to lay out the, the process of a diagonal electrophoresis experiment. So we know that the first step is going to be to cleave the peptides into fragments. So we want to be able to cleave the 2 peptides into fragments. And so, that will be the first step. And once we've cleaved them into fragments, we know that their disulfide bonds are going to remain intact. Wherever they are, they're going to remain intact. And so the second step, step number 2, is going to be to run SDS PAGE. And so when we run SDS PAGE, we're going to separate the fragments into, based on their molecular size. But again, the fragments where the disulfide bonds are included are going to migrate together because those fragments are going to be covalently linked. And so in the 3rd protein in the gel to performic acid, and we know that performic acid is going to cleave disulfide. So we can say performic acid to cleave disulfides. And once we've cleaved those disulfides, we can move on to our 4th step, which is going to be, of course, to run SDS PAGE in the perpendicular direction. And so, what that allows us to do is to separate out the fragments that were, disulfide linked, that contained disulfides. And so if the original and the mutant, protein generate the same exact pattern, in this second SDS PAGE gel, that would be reason to conclude that perhaps they have the same exact disulfide, bonds. But if they generated different fragments, then that would say that the mutant has a different, disulfide pairing than the original protein. And so, after we look at that band pattern in the second SDS PAGE gel, we can, of course, take our, separated fragments that lie off of the diagonal sequencing. So we can, isolate and sequence the disulfide linked peptides. And so, that would allow us to determine the actual positions of the disulfide bonds, which cysteine residues were formed, were forming those disulfides. And so this is a very brief layout of the diagonal electrophoresis experiment, but make sure that you're, you understand the general process of all of these steps. And so this concludes, the answer to our practice problem and I'll see you guys in our next video.

Struggling with Biochemistry?

Watch next

Diagonal Electrophoresis practice set