PROTEIN SEQUENCING

GUNJAN MEHTAVSC, RAJKOT

Why to determine Amino acid Sequence?:

This "amino acid analysis" tells us what the building blocks are in the peptide, but it tells us nothing about their sequence, the order in which they are joined. This information is lost when the peptide bonds which preserve that sequence are hydrolyzed. Even with as few as two amino acids, there are two possible sequences. Consider a dipeptide which amino acid analysis gives us gly and ala. Either of these could be the N terminus, so the dipeptide could be either gly-ala or ala-gly. Problem 18.6 in Brown gives you some experience with a pentapeptide, and things rapidly get more complex as the number of amino acid units in the peptide increases.

Key steps involved in process:

Knowing the ratio of amino acids Hydrolysis

Fragmenting the protein with proteases/any agent Knowing the number of diS bonds

Alkylation and reduction C-terminal end fixing with a solid support N-terminal end sequencing/Edman degradation

Determination of N- terminal:

N terminus determination is commonly done by a process called the Edman degradation. The chemistry is outlined as follows.

This reaction can be understood if we look for some analogies that will help us apply the patterns we used in the past. 1. The -N=C=S group resembles a CO 2 (O=C=O) molecule

in that the carbon atom is connected to two electronegative atoms by a double (sigma and pi) bond.

2. We know from reacting Grignard reagents with CO 2 that the nucleophile attacks the carbon in CO 2 , so we can expect the same type of pattern in the Edman degradation.

3. The nucleophile is the free NH 2 group at the N terminus of the peptide, formed by loss of a proton from the NH 3 +to some unspecified base.

4. As we have seen with other reactions of NH 2 groups, this step is followed by a proton shift.

5. The product of the addition of N and H to the C=N double bond has a nucleophilic sulfur atom located just in reach of the carbonyl carbon at the other end of the N terminal amino acid.

6. Attack of this sulfur at that carbonyl group is followed by departure of the NH group of the next amino acid.

7. This cleaves the peptide bond between the N terminal amino acid and the next amino acid.

8 . Further reshuffling of protons yields an isomer of the phenylthiohydantoin.

9. This isomer is converted to the phenylthiohydantoin during the treatment with HCl and the phenylthiohydantoin is identified.

10. Since the phenylthiohydantoin includes the R group of the N terminal amino acid, identification of the phenylthiohydantoin also identifies the N terminal amino acid.

Multimedia

Edman degradation animation