27.8 Introduction to Peptide Structure Determination

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. O O O + – protein H3NCHC NHCHCO C R R Carboxypeptidase Carboxypeptidase is a proteolytic enzyme(catalyzes the hydrolysis of proteins).

9. O O O + – protein H3NCHC NHCHCO C R R Carboxypeptidase Carboxypeptidase is a proteolytic enzyme(catalyzes the hydrolysis of proteins). Carboxypeptidase is selective for cleavingthe peptide bond to the C-terminal amino acid.

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

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

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 (Section 23.5).

15. NO2 O O O O – O2N NHCHCO F NHCHC NHCH2C H2NCHC 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 – 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. +

17. 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.

18. NO2 O O2N NHCHCOH CH(CH3)2 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. H3O+

19. NO2 O O O O + + H3NCHCO– H3NCH2CO– O2N NHCHCOH CH2C6H5 CH(CH3)2 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. + + + + H3NCHCO– CH3 H3O+

20. 27.12Insulin

21. 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.

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

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

24. 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

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

26. 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.

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

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

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

30. 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

31. 27.13The Edman Degradation and Automated Sequencing of Peptides

32. 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.

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

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

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

36. 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.

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

38. 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. +

39. 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. +