1. 27.8 Introduction 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. 27.9 Amino 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. 27.10 Partial 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. Carboxypeptidase protein H 3 NCHC O R + NHCHCO OR – CO Carboxypeptidase is a proteolytic enzyme (catalyzes the hydrolysis of proteins).

9. Carboxypeptidase protein H 3 NCHC O R + NHCHCO OR – CO Carboxypeptidase is a proteolytic enzyme (catalyzes the hydrolysis of proteins). Carboxypeptidase is selective for cleaving the peptide bond to the C-terminal amino acid.

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

11. Chymotrypsin Chymotrypsin is selective for cleaving the peptide bond to the carboxyl group of amino acids with an aromatic side chain. NHCHC O R' OR" O R phenylalanine, tyrosine, tryptophan

12. 27.11 End 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. 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). F O2NO2NO2NO2N NO 2

15. Sanger's Method 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid. F O2NO2NO2NO2N NO 2 NHCH 2 C NHCHCO CH 3 NHCHC CH 2 C 6 H 5 H 2 NCHC OOO O CH(CH 3 ) 2 – +

16. Sanger's Method 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid. F O2NO2NO2NO2N NO 2 NHCH 2 C NHCHCO CH 3 NHCHC CH 2 C 6 H 5 H 2 NCHC OOO O CH(CH 3 ) 2 – + O2NO2NO2NO2N NO 2 NHCH 2 C NHCHCO CH 3 NHCHC CH 2 C 6 H 5 NHCHC O O O O CH(CH 3 ) 2 –

17. 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. O2NO2NO2NO2N NO 2 NHCH 2 C NHCHCO CH 3 NHCHC CH 2 C 6 H 5 NHCHC O O O O CH(CH 3 ) 2 –

18. 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. O2NO2NO2NO2N NO 2 NHCH 2 C NHCHCO CH 3 NHCHC CH 2 C 6 H 5 NHCHC O O O O CH(CH 3 ) 2 – H3O+H3O+H3O+H3O+ O O2NO2NO2NO2N NO 2 NHCHCOH CH(CH 3 ) 2

19. 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. O2NO2NO2NO2N NO 2 NHCH 2 C NHCHCO CH 3 NHCHC CH 2 C 6 H 5 NHCHC O O O O CH(CH 3 ) 2 – H3O+H3O+H3O+H3O+ H 3 NCHCO – CH 3 + H 3 NCH 2 CO – O O O O2NO2NO2NO2N NO 2 NHCHCOH CH(CH 3 ) 2 +O H 3 NCHCO – CH 2 C 6 H 5 + + + +

20. 27.12 Insulin

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. 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. 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. 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. Primary Structure of Bovine Insulin N terminus of A chain N terminus of B chain C terminus of B chain C terminus of A chain 5 5 15 10 15 20 20 25 30SS10 S S S S F F F F V N Q HL C C C C C V V V V G G G S S S H L L L G A A A C L Y Y E E L E R Y Y I Q K P T Q N N

31. 27.13 The 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. Edman Degradation The key reagent in the Edman degradation is phenyl isothiocyanate. NC S

34. Edman Degradation Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid. peptide H 3 NCHC OR + NHNHNHNH C6H5NC6H5NC6H5NC6H5NC S +

35. Edman Degradation peptide H 3 NCHC OR + NHNHNHNH C6H5NC6H5NC6H5NC6H5NC S + peptide C 6 H 5 NHCNHCHC OR NHNHNHNHS

36. Edman Degradation peptide C 6 H 5 NHCNHCHC OR NHNHNHNHS The product is a phenylthiocarbamoyl (PTC) derivative. 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.

37. Edman Degradation peptide C 6 H 5 NHCNHCHC OR NHNHNHNHSHCl peptide H3NH3NH3NH3N++ C6H5NHC6H5NHC6H5NHC6H5NH CSC N CH R O

38. Edman Degradation C6H5NHC6H5NHC6H5NHC6H5NH CSC N CH R O The product is a thiazolone. Under the conditions of its formation, the thiazolone rearranges to a phenylthiohydantoin (PTH) derivative. peptide H3NH3NH3NH3N++

39. Edman Degradation C6H5NHC6H5NHC6H5NHC6H5NH CSC N CH R O C C N HN CH R OS C6H5C6H5C6H5C6H5 The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation. peptide H3NH3NH3NH3N++