Bio Q- Passage

[WhittyPsyche]

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Okay so the question, from a passage but not explicit, more memory

"To determine the primary structure of the protein portion of Photosystem I, a series of cleavage reactions was undertaken. To break apart the protein, the most logical action to take would be to:
A) decarboxylate free carboxyl groups
B) hydrolysis peptide bonds
C) repolymerize peptide bonds
D) hydrolyze amuse branch points"

Answer: B

Okay, please tell me if I am over thinking this. But to me this is a poorly put question. If you want to determine the primary structure, you wouldn't hydrolyze the peptide bonds (which hold the primary structure together). I mean hydrolyzing everything will give you free AAs.
I really need to know if these are poor answer choices or if I need to change the way I am interpreting the set up for the question.


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[theonlytycrane]

Okay, please tell me if I am over thinking this. But to me this is a poorly put question. If you want to determine the primary structure, you wouldn't hydrolyze the peptide bonds (which hold the primary structure together). I mean hydrolyzing everything will give you free AAs.
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Why did this answer choice bother you specifically?

We want to break apart a protein and proteins are held together via peptide bonds. (B) fits the question best- don't overthink 🙂

 

[FCMike11]

Why did this answer choice bother you specifically?

We want to break apart a protein and proteins are held together via peptide bonds. (B) fits the question best- don't overthink 🙂

Yeah, but his point is is that the primary structure is the order of amino acids. How can you evaluate the order of amino acids if after your treatment you just have free AAs? Unless I am missing something as well which would indicate what order they were linked.

 

[theonlytycrane]

@FCMike11 I understand the "point". I haven't seen the passage and am also not an expert at sequencing techniques. But you could hydrolyze the peptide chain amino acid by amino acid, purifying and analyzing each fragment to determine the primary structure.

Think about something analogous to DNA sequencing for peptides.

 

[FCMike11]

@FCMike11 I understand the "point". I haven't seen the passage and am also not an expert at sequencing techniques. But you could hydrolyze the peptide chain amino acid by amino acid, purifying and analyzing each fragment to determine the primary structure.

Think about something analogous to DNA sequencing for peptides.

Word.

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[WhittyPsyche]

Yeah, but his point is is that the primary structure is the order of amino acids. How can you evaluate the order of amino acids if after your treatment you just have free AAs? Unless I am missing something as well which would indicate what order they were linked.

"She" but yes exactly.

I could easily see an answer like that saying: this is wrong because we want to determine the AA sequence, so we should not hydrolyze the primary structure. I would have preferred an option that said breaking H-bonds and disulfide linkages etc. Not destroying to primary structure to free AAs


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[WhittyPsyche]

@FCMike11 I understand the "point". I haven't seen the passage and am also not an expert at sequencing techniques. But you could hydrolyze the peptide chain amino acid by amino acid, purifying and analyzing each fragment to determine the primary structure.

Think about something analogous to DNA sequencing for peptides.

The passage is about photosynthesis which is why I mentioned at the top that it's not an explicit question.

And I see your second point about amino acid by amino acid but that kind of specificity only works with "regions". Aka break up the carboxyl of end after Lys residue etc, and it would affect all of them. DNA isn't the same as it has to be unzipped.

Obviously they expected that answer to be sufficient, but ugh.


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[theonlytycrane]

I agree that answers can be a bit unsatisfying at times and you have a good point about regions. There probably isn't a way to cut up the protein amino acid by amino acid so cleanly as I described, but what we could do is subject our protein to multiple different proteases that cleave after specific regions. Then purify the fragments, identify what the fragments looked like after each run and try to piece together what the peptide sequence is composed of based on our clues.

If we use chymotrypsin, for example, and nothing gets cut up, we know that there likely aren't any aromatic residues.

 

[aldol16]

Okay, please tell me if I am over thinking this. But to me this is a poorly put question. If you want to determine the primary structure, you wouldn't hydrolyze the peptide bonds (which hold the primary structure together). I mean hydrolyzing everything will give you free AAs.

It's not poorly worded - it's perfectly worded because it's vague. It doesn't tell you how it hydrolyzes the peptide bonds. Even if you add a bunch of water, the bonds won't just spontaneously hydrolyze. A specific series of chemical steps are needed to do this and one can take advantage of this fact to do peptide sequencing via hydrolysis.

I agree that answers can be a bit unsatisfying at times and you have a good point about regions. There probably isn't a way to cut up the protein amino acid by amino acid so cleanly as I described, but what we could do is subject our protein to multiple different proteases that cleave after specific regions. Then purify the fragments, identify what the fragments looked like after each run and try to piece together what the peptide sequence is composed of based on our clues.

Edman degradation if you take "hydrolysis" loosely.

 

[WhittyPsyche]

It's not poorly worded - it's perfectly worded because it's vague. It doesn't tell you how it hydrolyzes the peptide bonds. Even if you add a bunch of water, the bonds won't just spontaneously hydrolyze. A specific series of chemical steps are needed to do this and one can take advantage of this fact to do peptide sequencing via hydrolysis.



Edman degradation if you take "hydrolysis" loosely.

You're right. It can be vague and that's where I didn't like it. I will need to keep that in mind. I'm always after the most specific answer.


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