You are sequencing a protein. Previous work indicates that this protein consists

Question

You are sequencing a protein. Previous work indicates that this protein consists of two polypeptide chains that are held together by a single disulfide bond. You perform two experiments and get the results indicated below. From this ‘experimental’ information. Determine the sequences of the two polypeptide chains and explain how you obtain this?
Experiment #1
In this experiment, you cleave the disulfide bond with performic acid, separate the two chains, and sequence them by treatment with the proteolytic enzymes trypsin and chymotrypsin, followed by Edman degradation to determine the sequences of the each of the resulting fragments. Through using an overlap-peptide type of method, you should be able to determine the original sequence of the two separate polypeptide chains

Trypsin Treatment

chain 1:

I
LK
NQR
HSFWK
FWPCDR

chain 2:

GAR
MPAK
LSYSK
LSCFGR
LAACNIYF

Chymotrpsin Treatment

chain 1:

W
W
HSF
PCDRI
KNQRLKF

chain 2:

F
GRGARLSY
MPAKLSCF
SKLAACNIY

Explanation / Answer

Umm. heres the answer first, but explaining this is gonna be near impossible, but ill give it a shot Chain 1: HSFWKNQRLKFWPCDRI Chain 2: MPAKLSCFGRGARLSYSKLAACNIYF Okay so the key is to separate out and identify where the cut is happening. Trypsin will always cut on the C terminal side of K, and R (unless P is following it). Chemotrypsin will always cut on the C terminal side of Y, W and F. Thats all you need to know. Edman degradation is simply a method of sequencing where you sequence by reading the N-terminal acid and then cleaving it and continuing till you have finished reading. In this problem, it means that they are using Edman degradation to read out the chains that you see. So For Chain 1: I will put an underscore "_" to represent the side that it is cut. Trypsin cleavage _I LK_ NQR_ HSFWK_ FWPCDR_ Chemotrypsin cleavage W_ W_ HSF_ _PCDRI KNQRLKF_ Heres an initial trick. Find the longest sequence with the underscore on the left side, and that is your starting point. Since there are no cuts possibly to the right, you know this is the end of that chain. Now you compare the _PCDRI to the trypsin cleavage and you see that it overlaps with the FWPCDR_ and _I so you know thats the same point. If you put it together, you get _FWPCDRI Now you go back to Chemotrypsin and look for overlap of the recently added _FW. You see here that there is a W, so now you need to find something that ends with F_ and theres actually 2 choices. If you test out both, you will see that HSF_ doesn't lead to anything when you overlap with Trypsin cleavages, but the KNQRLKF_ does and if you keep on going, you will find the sequence is what I listed above. For chain 2, do the same thing, identify the last section, which is _LAACNIYF Overlap with the chemotrypsin cleavages and you find the next section to be LSYSK then keep on going and you end up with the solution I obtained at the top. Sorry if explanation is rough. This is hard topic to explain on paper. Remember, the key is to identify the starting point which is the longest sequence with the "_" to the left side. Then work from right to left to identify the sequence. Hope it helps.

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.