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Protein sequencing
 

Protein sequencing

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    Protein sequencing Protein sequencing Presentation Transcript

    • Why to determine Amino acid Sequence?:This "amino acid analysis" tells us what the buildingblocks are in the peptide, but it tells us nothing abouttheir sequence, the order in which they are joined. Thisinformation is lost when the peptide bonds whichpreserve that sequence are hydrolyzed. Even with asfew as two amino acids, there are two possiblesequences. Consider a dipeptide which amino acidanalysis gives us gly and ala. Either of these could bethe N terminus, so the dipeptide could be either gly-alaor ala-gly. Problem 18.6 in Brown gives you someexperience with a pentapeptide, and things rapidly getmore complex as the number of amino acid units in thepeptide 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 someanalogies that will help us apply the patterns we used in thepast.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