1. Protein Purification and Analysis Day 4

2. Amino Acids, Peptides, and Proteins

8. Absorption of ultraviolet light by aromatic amino acids

9. Amino acids are “zwitterions” at pH 7

10. Titration of glutamate

11. Titration of histidine

12. Formation of a peptide bond

13. Ser – Gly – Tyr – Ala - Leu + NH 3 ----------SGYAL----------- COO -

14. Peptides can ionize Peptides can be charged

15. Proteins are a variety of sizes

16. Proteins can contain very different ratios of amino acids

17. 1º 2º 3º 4º Polypeptide chains fold upon themselves to form a unique three-dimensional structure. There are four defined levels of protein structure

18. 1. The three-dimensional structure of a protein is determined by its amino acid sequence. 2. The function of a protein depends on its structure. 3. Each protein has a unique structure. Chymotrypsin Proteins have a specific conformation, the spatial arrangement of atoms. Proteins in their functional, folded conformations are in their native state. Native conformations are not very stable. Glycine

19. Question: Is the structure of every protein totally different, or are there common themes? Answer: There are regular folding patterns of the peptide backbone present in most proteins. Examples: alpha helix beta sheet

20. Hydrogen bond between (1) C=O===H-N (4) Alpha Helix

21. Beta sheet structures Side chains of adjacent amino acids protrude in opposite directions

22. Beta sheet structures

23. Many regions of secondary structure are connected by  turns

24. Tertiary (3º) structure of proteins Proteins fold into a globular structure that excludes H 2 O from the interior. There is a systematic arrangement of amino acid side chains in proteins. In general: Nonpolar amino acids are in the interior. Val, Leu, Ile, Met, Phe Charged amino acids are on the surface. Arg, Lys, His, Asp, Glu Uncharged polar amino acids are on the surface or in the interior. Ser, Thr, Asn, Gln, Tyr, Trp

25. Quaternary (4º) structure of proteins Some proteins form aggregates of 2 or more subunits. Reasons for multiple subunits in a protein include: 1. Cooperativity between subunits of a protein may be an important part of a protein’s function. Example: hemoglobin binds oxygen cooperatively. 2. A protein’s catalytic function may require amino acids from each subunit. Example: active HMG-CoA reductase is a dimer. 3. To fulfill a specific function a protein may have to be too large to be synthesized as a single subunit. Example: groEL chaperonin has 14 subunits

26. Working With Proteins Experimental techniques for protein analysis and characterization

27. A cell contains many types of proteins In the lab we want to isolate a single protein for experiments We first grow cells or isolate tissues that contain the protein of interest We break open the cells (lysis) to produce a crude extract Use centrifugation to separate soluble from insoluble material We fractionate the protein mixture based on properties of individual proteins such as size, charge or solubility. By using a combination of fractionation procedures a protein can be isolated from all other contaminating proteins in a cell. This process is called protein purification.

28. Fractionation by relative solubility The procedure of ammonium sulfate (AS) precipitation is used to separate proteins on the basis of their relative solubilities. The solubility of proteins is lowered at higher salt concentrations This is called salting out. As the amount of AS is increased, more proteins precipitate. A protein chemist wants to determine where the protein of interest precipitates and other proteins do not (or vice-versa).

29. Column Chromatography

30. Ion Exchange Chromatography

31. Size-exclusion Chromatography

32. Affinity Chromatography

34. SDS binds to protein molecules. One SDS per two amino acids

35. SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE)

36. SDS-PAGE of a protein mixture

38. Native PAGE "Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS. While in SDS- PAGE the electrophoretic mobility of proteins depends primarily on their molecular mass, in native PAGE the mobility depends on both the protein's charge and its hydrodynamic size. The electric charge driving the electrophoresis is governed by the intrinsic charge on the protein at the pH of the running buffer. This charge will, of course, depend on the amino acid composition of the protein as well as post-translational modifications such as addition of sialic acids. Since the protein retains its folded conformation, its hydrodynamic size and mobility on the gel will also vary with the nature of this conformation (higher mobility for more compact conformations, lower for larger structures like oligomers). If native PAGE is carried out near neutral pH to avoid acid or alkaline denaturation, then it can be used to study conformation, self-association or aggregation, and the binding of other proteins or compounds. Native gels can be sensitive to a process that alters either the charge or the conformation of a protein, such as protein-protein or protein-ligand interactions It is possible to recover proteins in their native, active state.

39. Isoelectric focusing (IEF)

41. 2-D electrophoresis

43. Laboratory Experiment Goals of Lab: Purify a recombinant form of the enzyme catalase expressed in the bacterium Escherichia coli (E. coli). The enzyme produced is the form of catalase found naturally in the bacterium Listeria monocytogenes. The gene encoding L. monocytogenes catalase gene was fused to a circular plasmid DNA used to produce proteins in E. coli. IMAC (immobilized metal affinity chromatography) will be used for purification Histidine Imidazole