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Here is the question first, and all of its parts (then I will attempt to answer it and list my questions):
15. You are determining the amino acid sequence of a protein isolated in the lab. You have used two different proteases to cleave the protein. These are the fragments after cleavage (all written with N terminus at the left and C terminus at the right)
Protease I ////// Protease II
NMLCST ////// STFSTNSC
NST ////// STNPGPT
NPGPT ////// NPGPTNC
NSCST ////// STNSTNMLC
15 A. Give the names of the amino acids each protease cleaves between (2 pts.)
B. You sequence each of the fragments from the cleavage reactions. Give the name of the technique and list the steps involved that would allow you to obtain the amino acid sequence of each fragment. (5 points)
C. What is the sequence of the one letter amino acid codes in the original protein? (4 points)
D. Do you think this is a transmembrane protein? Explain your answer. (3 points)
E. Circle the amino acid residues that are likely to be phosphorylated, what general class of enzyme is responsible for phosphorylation reactions? (4 points)
F. What levels, if any, of protein structure would change if an arginine is inserted after the 14th amino acid in the protein? (3 points)
A) Protease I: Only between asparagine and threonine
Protease II: Between Serine and cysteine, serine and threonine, and asaparagine and cysteine? (is this possible? I came to this conclusion by just examining the amino acids at the end of the polypeptides...not sure if that's the way to do it?).
B) Edman Degradation:
1) purify your protein
2) Use proteases that cleave different bonds between amino acids to cleave into segments no longer than 50 AA long
3) Treat with PITC
4) Cleave with acid
5) AA analysis of segments
6) Piece together protein
C) This is the part that I'm pretty confused about...I'm not very good at little puzzles like these:
I got this far and got lost...and I'm pretty sure that it is not right anyway:
D) I can't really figure this part out without knowing the protein code, but I know that if it is a transmembrane protein it should have a large region containing hydrophobic/nonpolar amino acids so that it can anchor itself into the membrane
E) So neither my professor or my book goes over which amino acids can be phosphorylated...but after some research on the internet I've come up with: serine, threonine, tyrosine, and histidine? Is this right? And as for the enzyme... either kinase or phosphatase?
F) Again, I think I need the amino acid code for this, but I know that arginine is a positively charged polar molecule under physiologic conditions.
Thanks in advance
If I understand it correctly, peptides in one column originate from one peptidase, right? Can you check again, whether are they correct? For example for PI, the third and last peptides are the same. Further, first, fourth and fifth peptides contain CST, but there is always other amino acid before this. Should these peptides cover whole sequence?
Cis or trans? That's what matters.
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