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27.8 Introduction to Peptide Structure Determination

27.8 Introduction to Peptide Structure Determination. Primary Structure. The primary structure is the amino acid sequence plus any disulfide links. Classical Strategy (Sanger). 1. Determine what amino acids are present and their molar ratios.

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27.8 Introduction to Peptide Structure Determination

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  1. 27.8Introduction to Peptide Structure Determination

  2. Primary Structure • The primary structure is the amino acid sequence plus any disulfide links.

  3. Classical Strategy (Sanger) • 1. Determine what amino acids are present and their molar ratios. • 2. Cleave the peptide into smaller fragments, and determine the amino acid composition of these smaller fragments. • 3. Identify the N-terminus and C-terminus in the parent peptide and in each fragment. • 4. Organize the information so that the sequences of small fragments can be overlapped to reveal the full sequence.

  4. 27.9Amino Acid Analysis

  5. Amino Acid Analysis • Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture of amino acids. • The mixture is separated by ion-exchange chromatography, which depends on the differences in pI among the various amino acids. • Amino acids are detected using ninhydrin. • Automated method; requires only 10-5 to 10-7 g of peptide.

  6. 27.10Partial Hydrolysis of Proteins

  7. Partial Hydrolysis of Peptides and Proteins • Acid-hydrolysis of the peptide cleaves all of the peptide bonds. • Cleaving some, but not all, of the peptide bonds gives smaller fragments. • These smaller fragments are then separated and the amino acids present in each fragment determined. • Enzyme-catalyzed cleavage is the preferred method for partial hydrolysis.

  8. Partial Hydrolysis of Peptides and Proteins • The enzymes that catalyze the hydrolysis of peptide bonds are called peptidases, proteases, or proteolyticenzymes.

  9. O O O NHCHC NHCHC NHCHC R" R R' lysine or arginine Trypsin Trypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine.

  10. O O O NHCHC NHCHC NHCHC R" R R' phenylalanine, tyrosine, tryptophan Chymotrypsin Chymotrypsin is selective for cleaving the peptidebond to the carboxyl group of amino acids withan aromatic side chain.

  11. O O O + – protein H3NCHC NHCHCO C R R Carboxypeptidase Carboxypeptidase is selective for cleavingthe peptide bond to the C-terminal amino acid.

  12. 27.11End Group Analysis

  13. End Group Analysis • Amino sequence is ambiguous unless we know whether to read it left-to-right or right-to-left. • We need to know what the N-terminal and C-terminal amino acids are. • The C-terminal amino acid can be determined by carboxypeptidase-catalyzed hydrolysis. • Several chemical methods have been developed for identifying the N-terminus. They depend on the fact that the amino N at the terminus is more nucleophilic than any of the amide nitrogens.

  14. NO2 O2N F Sanger's Method • The key reagent in Sanger's method for identifying the N-terminus is 1-fluoro-2,4-dinitrobenzene. • 1-Fluoro-2,4-dinitrobenzene is very reactive toward nucleophilic aromatic substitution (Chapter 23).

  15. NO2 O O O O – O2N NHCHCO F NHCHC NHCH2C H2NCHC CH3 CH2C6H5 CH(CH3)2 NO2 O O O O – NHCHCO NHCHC NHCH2C O2N NHCHC CH3 CH2C6H5 CH(CH3)2 Sanger's Method • 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid. +

  16. NO2 O O O O + + + + + + H3NCHCO– H3NCHCO– H3NCH2CO– O2N NHCHCOH CH3 CH2C6H5 CH(CH3)2 H3O+ NO2 O O O O – NHCHCO NHCHC NHCH2C O2N NHCHC CH3 CH2C6H5 CH(CH3)2 Sanger's Method • Acid hydrolysis cleaves all of the peptide bonds leaving a mixture of amino acids, only one of which (the N-terminus) bears a 2,4-DNP group.

  17. 27.12Insulin

  18. Insulin • Insulin is a polypeptide with 51 amino acids. • It has two chains, called the A chain (21 amino acids) and the B chain (30 amino acids). • The following describes how the amino acid sequence of the B chain was determined.

  19. The B Chain of Bovine Insulin • Phenylalanine (F) is the N terminus. • Pepsin-catalyzed hydrolysis gave the four peptides:FVNQHLCGSHL VGAL VCGERGF YTPKA

  20. The B Chain of Bovine Insulin FVNQHLCGSHL VGAL VCGERGF YTPKA

  21. The B Chain of Bovine Insulin • Phenylalanine (F) is the N terminus. • Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA • Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC

  22. The B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALY YLVC VCGERGF YTPKA

  23. The B Chain of Bovine Insulin • Phenylalanine (F) is the N terminus. • Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA • Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC • Trypsin-catalyzed hydrolysis gave GFFYTPK which completes the sequence.

  24. The B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALY YLVC VCGERGF GFFYTPK YTPKA

  25. The B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALY YLVC VCGERGF GFFYTPK YTPKA FVNQHLCGSHLVGALYLVCGERGFFYTPKA

  26. Insulin • The sequence of the A chain was determined using the same strategy. • Establishing the disulfide links between cysteine residues completed the primary structure.

  27. S S 15 5 C L Y S Q E Q V C L I F E N 20 C V Y A S C S N 10 S S H L Q N V C S F G Y A L L G S H V V L 5 C 20 G 15 10 E R G Y T F F P K A 25 30 Primary Structure of Bovine Insulin N terminus of A chain C terminus of A chain N terminus of B chain C terminus of B chain

  28. 27.13The Edman Degradation and Automated Sequencing of Peptides

  29. Edman Degradation • 1. Method for determining N-terminal amino acid. • 2. Can be done sequentially one residue at a time on the same sample. Usually one can determine the first 20 or so amino acids from the N-terminus by this method. • 3. 10-10 g of sample is sufficient. • 4. Has been automated.

  30. N C S Edman Degradation • The key reagent in the Edman degradation is phenyl isothiocyanate.

  31. O + peptide NH H3NCHC C6H5N C S R Edman Degradation • Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid. +

  32. S O peptide C6H5NHCNHCHC NH R O + peptide NH H3NCHC C6H5N C S R Edman Degradation +

  33. S O peptide C6H5NHCNHCHC NH R Edman Degradation • The PTC derivative is then treated with HCl in an anhydrous solvent. The N-terminal amino acid is cleaved from the remainder of the peptide. The product is a phenylthiocarbamoyl (PTC)derivative.

  34. S O peptide C6H5NHCNHCHC NH R S O C C6H5NH C + peptide H3N CH N R Edman Degradation HCl +

  35. S O C C6H5NH C + peptide H3N CH N R Edman Degradation The product is a thiazolone. Under the conditions of its formation, the thiazolonerearranges to a phenylthiohydantoin (PTH) derivative. +

  36. C6H5 N S O C C CH HN R S O C C6H5NH C + peptide H3N CH N R Edman Degradation • The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation. +

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