I don't understand how to do this at all!! I've been Googling the past hour and I just don't get it. My teacher won't respond and my exam is tomorrow. I get that we cleave with an acid and then we can do amino acid analysis. The entire protein can be pieced together only after sequencing of two sets of protease fragments. Can someone help me or even just refer me to something that does help?
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
NMLCST NST
NPGPT
NSCST
NCSTFST
NPGPT
Protease II
STFSTNSC
STNPGPT
NPGPTNC
STNSTNMLC
A. Give the names of the amino acids each protease cleaves between (2 pts.)
Protease I:
Protease II:
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)
-- Edman degradation
C. What is the sequence of the one letter amino acid codes in the original protein? (4 points)
A. The amino acids each protease cleaves between are:
Protease I:
- NMLCST cleaves between amino acids N and M
- NST cleaves between amino acids T and N
- NPGPT cleaves between amino acids G and P
- NSCST cleaves between amino acids T and N
- NCSTFST cleaves between amino acids T and F
Protease II:
- STFSTNSC cleaves between amino acids S and T
- STNPGPT cleaves between amino acids G and P
- NPGPTNC cleaves between amino acids C and N
- STNSTNMLC cleaves between amino acids M and L
B. The technique that can be used to sequence each of the fragments from the cleavage reactions is called Edman degradation. The steps involved in obtaining the amino acid sequence of each fragment using Edman degradation are:
1. React the cleaved fragment with phenyl isothiocyanate (PITC) to derivatize the N-terminal amino acid.
2. Treat the derivatized fragment with trifluoroacetic acid (TFA) to cleave the N-terminal amino acid from the fragment.
3. Separate the released amino acid using high-performance liquid chromatography (HPLC).
4. Identify the released amino acid using the phenylthiohydantoin (PTH) derivative, which is specific for each amino acid.
5. Repeat the steps for each subsequent amino acid in the sequence, starting with the original fragment and using progressively shorter fragments obtained from further protease cleavages.
C. The sequence of the one-letter amino acid codes in the original protein, based on the given fragments, is:
NSTNPGPTNCSTFSTNSCSTNSTNMLC
A. To determine the names of the amino acids cleaved by each protease, we need to analyze the fragments provided.
For Protease I:
- NMLCST: The cleavage occurs between amino acids N and M.
- NST: No cleavage occurs here.
- NPGPT: No cleavage occurs here.
- NSCST: The cleavage occurs between amino acids S and C.
- NCSTFST: The cleavage occurs between amino acids T and F.
- NPGPT: No cleavage occurs here.
Therefore, Protease I cleaves between the following amino acids:
- Between N and M
- Between S and C
- Between T and F
For Protease II:
- STFSTNSC: The cleavage occurs between amino acids C and S.
- STNPGPT: No cleavage occurs here.
- NPGPTNC: The cleavage occurs between amino acids P and N.
- STNSTNMLC: The cleavage occurs between amino acids C and S.
Therefore, Protease II cleaves between the following amino acids:
- Between C and S
- Between P and N
- Between C and S
B. To sequence each fragment, we can use a technique called Edman degradation. Here are the steps involved:
1. First, we treat each fragment separately with a reagent called phenylisothiocyanate (PITC), which selectively attaches to the N-terminus of the fragment.
2. The PITC modification allows us to conduct the Edman degradation. In this process, the N-terminal amino acid is cleaved off from the fragment one by one.
3. The released amino acid is reacted with a suitable reagent, such as dinitrofluorobenzene, to form a colored derivative that can be detected.
4. After detection, the N-terminal amino acid is removed, and the process is repeated to sequence the next amino acid in the fragment.
5. This sequential removal of amino acids and detection allows us to identify the amino acid sequence of each fragment.
By following these steps, we can obtain the amino acid sequence of each protease fragment.
C. To determine the sequence of the one-letter amino acid codes in the original protein, we need to piece together the sequences of the fragments.
Based on the given protease cleavage sites and the corresponding fragment sequences, we can rearrange and overlap the fragments to obtain the complete sequence.
Taking the fragments from Protease I and Protease II, we can align them as follows:
[N]MLCSTQNPGPTNCSTFSTNSC[T]NSTNMLC
Through this alignment, we get the complete sequence:
NMLCSTQNPGPTNCSTFSTNSCNSTNMLC
Therefore, the sequence of the one-letter amino acid codes in the original protein is:
N-M-L-C-S-T-Q-N-P-G-P-T-N-C-S-T-F-S-T-N-S-C-N-S-T-N-M-L-C