BCH 4053--General Biochemistry I--Spring 2003

Extra Credit Problem #2

This is to be done as a group project. At least two persons must work together, but three or more is okay. The problem is worth six points as follows:

1 point: attempt to answer
2 points: make a reasonable start
3 points: get some of the sequence
4 points: get majority of the sequence
5 points: most of sequence correct, one or two minor errors
6 points: correct sequence determined.

The problem is due in class on Friday, February 14. Please make your submission legible and in pencil or pen dark enough for me to read.

SEQUENCING WITHOUT SEPARATING PEPTIDES

To save time in sequencing, sometimes the Edman degradation procedure can be run on a mixture of peptides without first separating them. The results are like a jigsaw puzzle which one can then piece together to get the overall sequence. The following data are sufficient to deduce a complete sequence for the small protein described.

1. A small protein, containing only 40 amino acid residues was reduced and carboxymethylated to alkylate the cysteine residues and then characterized as follows:

(a) Edman degradation gave Asp as N-terminal.

(b) CNBr cleavage, followed by Edman sequencing of the unfractionated mixture of peptides gave the following sets of amino acids in each round of sequencing (each set listed in alphabetical order; data beyond the 8th round were not reliable):

Round

 

1
2
3
4
5
6
7
8
Amino Acids: Arg Gln Asn Arg Asn Arg Ala Ala
  Asp Pro Pro His Ilu His Gly Lys
  Glu Thr Ser Ilu Leu Trp Phe Met
  Gly Tyr Tyr Val Phe Val Thr Tyr

Note: This means that in the first round of the Edman, Arg, Asp, Glu and Gly were released, indicating four peptides with these amino acids as N-terminal. On the next round, Gln, Pro, Thr, and Tyr were released, etc.

(c) Trypsin digestion followed by Edman sequencing of the unfractionated mixture of peptides gave the following sets of amino acids in each round of sequencing (each set listed in alphabetical order; data beyond the 8th round were not reliable.): (Cys indicates carboxymethylcysteine.)

Round

 

1 2 3 4 5 6 7 8
Amino Acids: Asp Cys His Ala Ilu Arg Cys Glu
  Gly His Met Asn Leu Phe Lys Leu
  Gly Pro Thr Glu Thr Ser Ilu  
  Phe Pro Tyr Val Trp Ser    
  Tyr Tyr            

Note this time there were five peptides, one of them only two amino acids long, one of them six long, one seven, and two eight or longer.

Deduce the sequence of this 40-residue peptide from this information.

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