Protein Sequencing Problems

1) A small peptide was completely hydrolyzed to yield the following set of amino acids;
Ala + Arg + Gly + 2 Met + Lys + Ser + Val

In addition the peptide was subject to the following tests with the results given.

Sanger Reagent - DNP Gly and ,-DNP Lys

Carboxypeptidase A - a) Ser
b) All the rest
Cyanobromide - a) Ala + Gly + Lys + Met
b) Met + Val
c) Arg + Ser

Chymotrypsin - Nothing Released

Trypsin - a) Ala + Gly + Lys

b) Arg + 2 Met + Val
c) Ser

What is the order of amino acids in the peptide chain?

2) A small protein was completely hydrolyzed and found the contain Arg + Cys + 2 Lys
+ Met + Thr + Val. Based on the observations below, reconstruct the protein sequence.

FDNB - DNP-Arg released

Carboxypeptidase A a) Val
b) All the rest

Carboxypeptidase B Nothing released

Cyanobromide - a) Lys-Cys-Met-Arg
b) Thr-Val-Lys

Trypsin - a) Arg
b) Lys
c) Cys-Thr- Lys-Met
d) Val

Are there any unresolved amino acids? Which are they?

Answer Key

Problem #1

Sanger Gly must have been the AA on the far left side (amino side) of the protein
sequence. Lys has an NH2 on its side chain. That is why FDNB attacked it.

Chain = Gly ~

Carb A Carb A attacks the AA on the other end of the protein (the carboxylic acid
side). Since Ser was released, it must have been the last AA in the chain.

Chain = Gly ~~~~~~~~~~~~~~~~~~~~~~~~~~Ser

Cyanobromide This cleaves on the carboxyl side of Met. This means that anything
attached to the NH2 side of Met remains attached. There from each piece we deduce,

a) Gly is the first AA in the chain - Gly~(Ala~Lys)~ Met or Gly~( Lys~Ala)~ Met
b) Val~Met in this order because Val is attached to Mets NH2
c) Ser is the last AA so must be Arg~Ser

Chain = Gly ~(Ala~Lys)~Met~Val~Met~Arg~Ser or

Gly ~(Lys~Ala)~Met~Val~Met~Arg~Ser

Trypsin All we need is to resolve the position of Ala with respect to Lys. The
first piece cleaved off does this for us. If the order had been Gly~Lys~Ala~ then trypsin
would have cleaved between Lys and Ala and you would have gotten a piece containing
just Gly+Lys, but you didnt, you got Gly+Lys+Ala which means that the order must
have been Gly~Ala~Lys. So the order of the protein must have been,

Final Chain = Gly~Ala~Lys~Met~Val~Met~Arg~Ser

There are no unresolved amino acids

Problem #2

Moving a little faster this time,

Sanger/CarbA&B Gives us the following information

Chain = Arg~~~~~~~~~~~~~~Val

Cyano Cleaves Met on the right, so

a) Arg~(Lys~Cys)~Met or Arg~(Cys~Lys)~Met
b) (Thr~Lys)~Val or (Lys~Thr)~Val

Chain = Arg~(Lys~Cys)~Met~(Thr~Lys)~Val

Trypsin Cleaves Lys and Arg on the right, so

a) Arg and Lys were released, but since we also know Arg~(Lys~Cys)~Met from above,
the order must be Arg~Lys~Cys~Met

b&c) Since Val was released separately, Lys must have been attached to it, therefore
since we knew from Cyano (b) (Thr~Lys)~Val or (Lys~Thr)~Val, the order must have
been Thr~Lys~Val.

Final Chain = Arg~Lys~Cys~Met~Thr~Lys~Val

There are no unresolved amino acids.