1 / 33

Lecture 5

Lecture 5. Peptides : Primary sequence and experimental techniques. Peptide Bond. Peptide Bond Formation. Activated Intermediates. Naming Peptides. Levels of Protein Structure. Primary structure: Determination of sequence. Protein Homology. BLOSUM60 Matrix.

jayme
Download Presentation

Lecture 5

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Lecture 5 Peptides : Primary sequence and experimental techniques

  2. Peptide Bond

  3. Peptide Bond Formation Activated Intermediates

  4. Naming Peptides

  5. Levels of Protein Structure Primary structure: Determination of sequence

  6. Protein Homology BLOSUM60 Matrix

  7. Disulfide bonds are part of the primarystructure Disulfide Bonds

  8. How to synthesize a peptide Solid phase synthesis: Merrifield synthesis 1 polypeptide of a determined length

  9. Peptide Bond Formation In Vitro 1. Attachment to of C-terminal amino acid (protected on amino group) to resin 2. Removal of protecting group 3. Activation of next amino acid 4. Peptide bond formation 5. Removal of polypeptide from resin 6. Purification of polypeptide

  10. Step 1: Attachment to Resin t-Boc Amino acid t-Boc

  11. Step 2: Remove Protecting Group

  12. Step 3: Amino Acid Activation

  13. Step 4: Peptide Bond Formation

  14. Step 5: Removal of Polypeptide HF

  15. How to determine 1º sequence 1.Determine amino acid composition 2. Determine identity of N-terminal amino acid(s) 3. For large proteins, reduce to conveniently sized peptides -Proteases -Chemical reagents 3a. Cleave disulfide bonds

  16. Overview of Protein Sequencing 3b. Subject each peptide to stepwise removal and identification of amino acids from one end -Edman chemistry 4. Determine order of peptides in original protein and location of disulfide bonds

  17. Amino acid composition Hydrolyze protein with 6M HCl Separate amino acids via column chromatography Quantify using ninhydrin reaction

  18. Reactions • Used for detection and quantitation Ninhydrin reaction with amino acids

  19. Reactions Fluorescamine Reaction Highly Fluorescent

  20. Determination of N-Terminus: Sanger

  21. NO 2 O N N 2 H NO 2 Free Gly and Leu O N N 2 H Determination of N-Terminus: Sanger O Ala Gly Leu C O Strong Acid + Ala

  22. O H N Ala Gly Leu C O 3 Similarreactions can be done with dansyl chloride and dabsyl chloride CH3 SO2Cl N N N CH3 1.Reaction 2.Hydrolysis + CH3 SO2-NH-Ala N + other amino acids N N CH3 Highly fluorescent:good for very small quantities of protein

  23. Cleaving Disulfide Bonds Reduction and alkylation or oxidation

  24. Cleaving Disulfide Bonds Reduction:

  25. S H CH O- 2 + HI CH 2 Cleaving Disulfide Bonds O Acetylation: + Cys I C O O- S Cys C

  26. Oxidation of Disulfide Bonds

  27. Sequencing with Edman Degradation

  28. Sequencing with Edman Degradation (mildly acidic) (remaining peptide)

  29. H N N H Trypsin 2 Lys, Arg Chymotrypsin 2 Tyr, Trp, Phe Pepsin 1 Leu, Phe, Trp, Tyr Elastase 2 Ala, Gly, Ser, Val Cutting Large Proteins with Proteases (Table 3-7) O R CH C C 1 2 O _______R_______ (Others slowly)

  30. Peptide Cleavage with CNBr

  31. Peptide Cleavage with CNBr

  32. Protein Cleavage with CNBr

  33. Sequencing a large peptide:

More Related