1. Chapter 3.2 and 3.3: Peptides, Proteins, and Working with Proteins CHEM 7784 Biochemistry Professor Bensley

2. CHAPTER 3.2 and 3.3 Peptides, Proteins, and Working with Proteins The structure and properties of peptides The ionization behavior of and peptides Various methods to characterize peptides and proteins Today’s Objectives: to understand

3. Formation of Peptides Peptides are small condensation products of amino acids They are “small” compared to proteins (M w < 10 kDa)

4. Peptide Ends are Not the Same Numbering starts from the amino terminus AA 1 AA 2 AA 3 AA 4 AA 5

5. The Three Letter Code Naming starts from the N-terminus Sequence is written as: Ala-Glu-Gly-Lys Sometimes the one-letter code is used: AEGK

6. Peptides: A Variety of Functions Hormones and pheromones Neuropeptides Antibiotics Protection, e.g. toxins

7. Proteins are: Polypeptides (covalently linked  -amino acids) + possibly: cofactors coenzymes prosthetic groups other modifications

8. Polypeptide Size in Some Proteins

9. Classes of Conjugated Proteins

10. What to Study about Peptides and Proteins? What is its sequence and composition? What is its three-dimensional structure? How does it find its native fold? How does it achieve its biochemical role? How is its function regulated? How does it interact with other macromolecules? How is it related to other proteins? Where is it localized within the cell? What are its physico-chemical properties?

11. A Mixture of Proteins Can Be Separated Separation relies on differences in physico-chemical properties –Charge –Size –Affinity for a ligand –Solubility –Hydrophobicity –Thermal stability Chromatography is commonly used for preparative separation

12. Column Chromatography

13. Separation by Charge

14. Separation by Size

15. Separation by Affinity

16. Electrophoresis for Protein Analysis Separation in analytical scale is commonly done by electrophoresis –Electric field pulls proteins according to their charge –Gel matrix hinders mobility of proteins according to their size and shape

18. SDS PAGE: Molecular Weight SDS – sodium dodecyl sulfate – a detergent SDS micelles bind to, and unfold, all the proteins –SDS gives all proteins an uniformly negative charge –The native shape of proteins does not matter –Rate of movement will only depend on size: small proteins will move faster