In: Biology
The ubiquitin protein from the malaria parasite was treated with chymotrypsin, and the resulting fragments were sequenced. A second sample of the polypeptide was treated with trypsin, and the fragments were sequenced. What is the sequence of the polypeptide?
Chymotrypsin fragments:
AGKQLEDGRTLSDY
IPPDQQRLIF
VKTLTGKTITLDVEPSDTEIN-VKAKIQDKEGI
NIQKESTLHLVLRLRGGMQIF
Trypsin Fragments:
LR
AK
EGI
IQDK
LIFAGK
QLEDGR
TLTGK
IPPDQQR
GGMQIFVK
TLSDYNIQK
ESTLHLVLR
The sequence is
IPPDQQRLIF AGKQLEDGRTLSDY NIQKESTLHLVLRLRGGMQIF VKTLTGKTITLDVEPSDTEIN-VKAKIQDKEGI
Chymotrypsin cleavage site is
NH-R-R'-CO
R= phenylalanine (F), Tyrosine (Y), tryptophan (W)
R' is not proline (P)
The cleavage site of trypsin is
NH-R-CO
R= Lysine (K) or arginine (R)
In both the digestion we can see a fragment snding with Isoleucine
(I). It is not a cleavage site of both the enzymes. Hence it is
considered as the carboxy terminal of the polypeptide. Using this
fragment (VKTLTGKTITLDVEPSDTEIN-VKAKIQDKEGI) we shall predict the
sequence.
We shall consider the first 2 amino acid "VK" in
VKTLTGKTITLDVEPSDTEIN-VKAKIQDKEGI for our analysis. Look for
sequence ending with VK in trypsin.
We have GGMQIFVK. Now look for this sequence QIF in carboxy
terminus of chymotrypsin fragments. We have this
NIQKESTLHLVLRLRGGMQIF. Hence this fragment will come before the
terminal sequence.
NIQKESTLHLVLRLRGGMQIF VKTLTGKTITLDVEPSDTEIN-VKAKIQDKEGI
Again look for NIQK in the trypsin fragment. We have TLSDYNIQK. Look for TLSDY in chymotrypsin fragments. We have AGKQLEDGRTLSDY.
The sequence becomes
AGKQLEDGRTLSDY NIQKESTLHLVLRLRGGMQIF
VKTLTGKTITLDVEPSDTEIN-VKAKIQDKEGI
Finally look for AGK in trypsin fragments and it is LIFAGK. Look for LIF in chymotrypsin fragments and it is IPPDQQRLIF.
Hence the answer is IPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLDVEPSDTEIN-VKAKIQDKEGI