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Offline MedStudent

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strange peptide assignment - who can help?
« on: August 15, 2015, 05:31:14 AM »
Hello Ladies and Gentlemen,

I have some questions concerning the following assignment and actually I am looking for a comfortable and safe way to solve exercises like that. I have to translate the exercise from German, so if it is confusing to understand, please do not hesitate to ask me, so I can clarify what is meant.

Alright here we go:
Quote
The complete hydrolysis of a paptide resulted in: Thr, 2 Cys, Arg, Asp, Met, Phe, Val, 2 Ile, Gly und NH4+

The Edman degradation resulted in PTH-Thr and PTH-Ile

The Carboxypeptidase separated Ile and Val

The reduction with β-Mercaptoethanol resulted in two peptides, which showed (after hydrolysis) the following constituents:
A: Met, Cys, Thr, Ile, Asp, Phe, Gly und NH4+
B: Arg, Ile, Val und Cys

Treating A with Chymotrypsin you get two peptides, one of the is: Thr, Phe, Asp und NH4+

The treatment of A with BrCN gives you a dipeptide: Cys and Ile

The treatment of B with Trypsin eventuates in Val.

Please name the structure of the peptide.


Ok, so from what I understand we have to put all these small peptides into the right order. But how to do that? The good news is, that I have the solution for this:

Quote
   ....N-Thr-Asn-Phe-Gly-Met-Cys-Ile-C
                                            |
                                            S
                                            |                                                     
                                            S
                                            |
                                   N-Ile-Cys-Arg-Val-C

But how to get there? The Edman degradation splits at the N-terminus, and the Carboxypeptidase at the C-terminus. So this would represent the first and the last amino acid. But how to go on from here? I would very much appreciate your help, especially because I didn't find anybody in Germany who could explain this to me ;-)

Offline Irlanur

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Re: strange peptide assignment - who can help?
« Reply #1 on: August 15, 2015, 07:22:15 AM »
Did you know what all these reagents acutally do? I guess you had it in a lecture or at least a script...?

Offline MedStudent

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Re: strange peptide assignment - who can help?
« Reply #2 on: August 15, 2015, 08:53:46 AM »
Hi,

first of all thanks for your reply. Well, we only had a general lecture on enzymes and that's pretty much all. Maybe we manage to figure out how to solve this together.
So let me clarify, that this stuff is obviously about a large peptide that underwent some changes using a variety of enzymes.

As I said: The Edman degradation splits at the N-terminus, and the Carboxypeptidase at the C-terminus.

Chymotrypsin splits preferably behind Aromatic amino acids.

"Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine" (see Wiki) --> basic amino acids

β-Mercaptoethanol --> here I think the reduction splits the disulfide bond between the two Cys and looking at the solution this makes sense, so we basically have two  peptides that are linked through the Cysteine.

Cyanogen bromide = BrCN splits selectively at the C-terminal-end after Methionine.

This is what we know about the enzymes/reagents and now we have to find a way to bring the amino acids of the peptides into the right sequence.

Fortunately we do have the solution, so this may help us here a bit.

Anyway, thanks for reading through it. Would be wonderful if you guys could help with this problem  :). If you have any questions or don't understand something of what i have written, just ask me. I promise that I am going to answer fast (and please oversee my spelling and grammar mistakes)

« Last Edit: August 15, 2015, 09:15:39 AM by MedStudent »

Offline Irlanur

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Re: strange peptide assignment - who can help?
« Reply #3 on: August 15, 2015, 11:05:26 AM »
So you have the basic building blocks. Now start solving the puzzle (maybe even physically create the building blocks).

The reduction with mercaptoethanol might be a good start, this gives you two peptides connected by disulfide bond. now start to assemble it, e.g. you know the N and c termini.

go on. it's just like being a detective.

Offline MedStudent

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Re: strange peptide assignment - who can help?
« Reply #4 on: August 15, 2015, 01:36:33 PM »
Thank you, I think that now I found a way to deal with this type of question. As you said, it is really the best way to start with the β-Mercaptoethanol and then to mud through the rest of the enzymes and peptides.

So here that means:

A: N - Met, Cys, Thr, Ile, Asp, Phe, Gly - C
B: N- Arg, Ile, Val und Cys -C

Firstly, I deal with A:
Edman tells us, which AA (amino acid, just to make it short) is the first from the N-terminal ending, in this case it's Ile for A.

1. Thr-Met-Cys-Ile-Asp-Phe-Gly

Now the Carboxypeptidase shows us the last AA, which is Ile

2. Thr-Met-Cys-Asp-Phe-Gly-Ile

BrCN cuts right after Met and the AA sequence is Cys-Ile, so we must have Met-Cys-Ile (Ile is also our last AA)

3. Thr-Asp-Phe-Gly-Met-Cys-Ile

The Chymotrypsine doesn't change much any more, but anyway our first three AA comply with the fact that Chymotrypsine gives us a peptide that consists of Thy, Phe, Asp.
In the solution they have Asn instead of Asp, but I think it's just a misspelling. So this would be the solution for A.

For B there is a quite similar procedure:

B: Arg, Ile, Val und Cys

Edmann + Caboxypeptidase give us the first and the last AA:

1. Ile-Arg-Cys-Val

Trypsin gives us Val AFTER an lysine or arginine, here we have Arg. Val stays at the C-end (just sayin' because first it thought to place Val just behind Arg, like Ile-Arg-Val-Cys, but that's wrong as Val has to be the last AA (Carboxypeptidase). So the right way would be:

2. Ile-Cys-Arg-Val


After that you have to put in the disulfide bond between the two Cys. Woohoo! Still, I would say this type of question leaves a lot of room to do mistakes, especially during a written exam. So after all, it s****  :)

And thank you Irlanur for putting me on the right track!

Offline Babcock_Hall

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Re: strange peptide assignment - who can help?
« Reply #5 on: August 15, 2015, 03:26:04 PM »
How are Asn and Asp different?  You may recall that ammonium ion was seen in one step.

Offline MedStudent

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Re: strange peptide assignment - who can help?
« Reply #6 on: August 15, 2015, 05:21:21 PM »
Hello,
thanks a lot for your reply!!!
So you're saying that in fact Asn is right because they added that ammonium ion to Asp? Looking at the first sentence of the exercise it seems you are completely right:
"The complete hydrolysis of a paptide resulted in: Thr, 2 Cys, Arg, Asp, Met, Phe, Val, 2 Ile, Gly and NH4+"

Do I have to add the NH4+ to the Asp replacing its OH?


And what about the rest of my solution? I have tried it with another similar assignment and it actually works, apart from that Asn/Asp thing. Is there a chronological order that you would recommend regarding the enzymes?
The best way is obviously to begin with the β-Mercaptoethanol and then to go on with Edman and Carboxypeptidase. Then BrCN, then Chymotrypsine. But I dont know if we can generalize it.
What do you think?

Offline Babcock_Hall

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Re: strange peptide assignment - who can help?
« Reply #7 on: August 17, 2015, 09:02:39 AM »
In asparagine the side chain is an amide (it might help to look at the structure).  What will happen to this amide under conditions that completely hydrolyze a peptide bond?

When solving problems of this type, I find it helpful to label the trypsin peptides T1, T2, etc, where 1 and 2 are arbitrary.  Then I write out a number of blanks that is equal to the number of residues in the peptide.  Then I start filling in the blanks.  Then I do the same for chymotrypsin, etc.

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