Protein Sequencing Problems To get you started, here is a table with the specificities for some of the enzymatic

and chemical procedures used in sequencing proteins: Procedure Edman Degradation Carboxypeptidase A Site C side of N terminus N side of C terminus N side of C terminus C side of Rn C side of Rn C side of Rn Specificity Rn = any amino acid Rn Arg, Lys, Pro Rn-1 Pro Rn Arg, Lys, AECys Rn-1 Pro Rn = Met Rn = Met, Trp Rn = Lys, Arg, AECys Rn+1 Pro Rn = Phe, Trp, Tyr, Leu Rn+1 Pro Rn = Leu, Ile, Phe, Trp, Tyr, Val Rn+1 Pro Rn = Leu, Asp, Glu, Phe, Tyr, Trp Rn+1 Pro Rn = Asp, Glu Comment Ineffective for blocked N termini Removes 1 (usually up to 4) residues sequentially Removes 1 (usually up to 4) residues senquentially Highly Specific Highly Specific Highly Specific

Carboxypeptidase B

Cyanogen Bromide Anhydrous Cyanogen Bromide Trypsin

Chymotrypsin

C side of Rn

Sometimes cleaves at other sites Sometimes cleaves at other sites Fairly nonspecific

Thermolysin

N side of Rn

Pepsin

N side of Rn

Acid Phosphatase

C side of Rn

Highly Specific

You have likely noticed that the table uses terms like Rn+1. Ive drawn a short piece of polypeptide so you can visualize where these cleavages are taking place.

Rn-1 N H
H N O

O N H

Rn+1

Rn

In this class, if the amino acids are listed with dashes connecting them, they are in sequence. If the amino acids are listed separated by commas, they are not in order.

For example.. If I treat Gly-Lys-Ala-Val-Met with trypsin I will get two pieces. One will be (Gly-Lys) and the other will be (Ala-Val-Met) If I only know the composition (and not the actual sequence for these pieces) it would be written (Gly, Lys) and (Ala, Met, Val).

Practice Problems
1. Lets start by determining the products we would get from treating a polypeptide with some of the procedures above. Using the polypeptide Ala-Arg-Asp-Lys-Pro-Cys-Phe-Met-Pro-Asn-Gly

A) What would acid hydrolysis give you?

B) What would you get if you used Chymotrypsin?

C) What would you get if you used Cyanogen bromide?

D) What would you get if you used Trypsin?

E) How about using Trypsin after aminoethylation?

2. OK, heres a short sequencing problem. You have a hexapeptide which gave the following experimental results. What is the sequence of the polypeptide? Acid Hydrolysis (Arg, Glu, Gly, Met, Phe, Trp) Trypsin (Arg, Glu, Phe) and (Gly, Met, Trp) CNBr (Gly, Trp) and (Arg, Glu, Met, Phe) Edman Degradation (Glu) Carboxypeptidase (Trp)

3. Now try a longer sequencing problem. You have a 11 amino acid polypeptide. Using the experimental results below try to determine as much of the sequence as you can. Gln-His-Ser-Tyr-Cys-Ser-Glu-Lys-Met-Thr-Asp-Ala Acid Hydrolysis (Ala, Asp, Cys, Glu, Glu, Met, Ser, Ser, Thr, Tyr) Edman Degradation Gln Carboxypeptidase Ala CNBr (Ala, Asp, Thr) (Cys, Gln, Glu, Lys, Met, Ser, Ser, Tyr) Trypsin (Ala, Asp, Met, Thr) (Cys, Gln, Glu, Lys, Ser, Ser, Tyr) Aminoethylation followed by trypsin (Glu, Lys, Ser) (Ala, Asp, Met, Thr) (AECys, Gln, Ser, Tyr)

Have you got the sequence? No? You should have a few ambiguous places left. Look at the amino acids you have left to place in order and consider the table I gave you on the first page. What experiment could you do to place the last few amino acids?

If you answered correctly, the fragments you would get are: (Ala, Asp) (Gln, Ser) (Cys, Ser, Tyr) (Glu, Lys, Met, Thr) Now you can finish the sequence:

4. When polypeptides contain disulfide bonds we can determine their location by looking at the cleavage patterns of the native (containing disulfide bonds) protein and comparing it to the cleavage patterns for the denatured (disulfide bonds broken) protein. In this example we will cleave the disulfide bonds using mercaptoethanol. Native Protein Data Acid hydrolysis (Arg, Asp, Cys, Cys, Cys, Gly, Lys, Met, Thr, Val) Trypsin (Arg, Thr) (Cys, Gly, Met) (Asp, Cys, Cys, Lys, Val) CNBr (Cys, Gly) (Arg, Asp, Cys, Cys, Lys, Met, Thr, Val) Edman (Val) and (Thr) Carboxypeptidase (Gly) and (Asp)

After treatment with mercaptoethanol Acid hydrolysis (Ala, Arg, Asp, Cys, Cys, Cys, Gly, Lys, Met, Thr, Val)

Trypsin (Arg, Thr) (Asp, Cys) (Cys, Gly, Met) (Cys, Lys, Val)

CNBr (Cys, Gly) (Arg, Asp, Cys, Thr) (Cys, Lys, Met, Val)

5. Not all polypeptides are linear. Some are circular. Determine the sequence for this circular decapeptide.

Acid Hydrolysis (Ala, Arg, Asp, Cys, Met, Met, Phe, Ser, Trp, Val) Trypsin (Ala, Arg, Asp, Cys, Met, Met, Phe, Ser, Trp Val) Aminoethylation followed by Trypsin (Ala, AECys, Met, Phe, Val) (Arg, Asp, Met, Ser, Trp) Chymotrypsin (Ala, Cys, Met, Trp, Val) (Arg, Asp, Met, Phe, Ser) CNBr (Ala, Arg, Met, Phe, Ser) (Asp, Cys, Met, Trp, Val)