Ionic properties of amino acids impart ionic properties to proteins

1. Ionic properties of amino acids impart ionic properties to proteins • in general these are SURFACE properties (i.e. charged sidechains are on solvent-exposed outside of folded structure) • affect protein-ligand binding (e.g. DNA-binding proteins) or catalysis • average charge on protein is an important consideration in the design of a purification process

2. pKa3 pKa2 pKa1

3. pI= isoelectric point--the pH at which the net charge is 0. For amino acids with no ionizable functional group, pI = (pK1 + pK2)/2 For amino acids with an ionizable functional group, the average of the 2 pKs surrounding the isoelectric form of the amino acid determines the pI.

4. pKa3 The isoelectric form of asp occurs between pK1 and pK2. pKa2 pI = 2.0 + 3.9 2 = 2.95 pKa1

6. Other Properties of Amino Acids • Stereochemistry (all biosynthetic proteins made up of L-isomer) • Hydropathy (partitioning between polar and nonpolar solvents as indicator of polarity) (see Table 6-2 in VVP p 150; Take Note p58) these two properties are major determinants of peptide conformation

8. See VVP Fig 4-3

10. VVP Fig 6-3 p 126

12. Example of a protein sequence MANSKINKQL DKLPENLRLN GRTPSGKLRS FVCEVCTRAF ARQEHLKRHY RSHTNEKPYP CGLCNRCFTR RDLLIRHAQK IDSGNLGETI SHTKKVSRTI TKARKNSASS VKFQTPTYGT PDNGGSGGTV LSEGEWQLVL HVWAKVEADV AGHGQDILIR LFKSHPETLE KFDRFKHLKT EAEMKASEDL KKHGVTVLTA LGAILKKKGH HEAELKPLAQ SHATKHKIPI KYLEFISEAI IHVLHSRHPG DFGADAQGAM NKALELFRKD IAAKYKELGY G N-terminus C-terminus

13. VVP page 150 “nonpolar” “polar”

15. VVP Fig 6-1 p 125

16. VVP Fig 5-1 p 94 C-termini N-termini

17. (Rasmol)

18. VVP Fig. 5-1 Bovine Insulin

19. VVP Fig. 5-3 ELISA

20. VVP Fig. 5-4 Salting Out

21. VVP Fig. 5-5 Ion Exchange Chromatography

22. VVP Fig. 5-6 Gel Filtration (SEC)

23. VVP Fig. 5-7 Affinity Chromatography

24. VVP Fug, 5-8 Purification of Stapylococcal Nuclease

25. Stryer Fig. 4.7 PAGE

26. Animation http://www.whfreeman.com/lodish/con_index.htm?03 choose animations and then SDS gel electrophoresis

27. Stryer Fig. 4.9 Coomassie blue stained SDS gel.

28. VVP Fig. 5-9 PAGE

29. Stryer Fig. 4.8

30. VVP Fig. 5-10 SDS-PAGE of Salmonella proteins

31. VVP Fig. 5-10 MW vs. Mobility in SDS-PAGE

32. Stryer Fig. 4.36 Western blot.

34. VVP Fig. 5-11 Density Gradient Ultracentrifugation

35. Stryer Fig. 4.14

36. Stryer Fig. 4.6 MALDI-TOF Matrix-assisted laser desroption-ionization- Time of Flight

37. Stryer Fig. 4.17

38. Determining Primary Structure 1. Determine aa composition

39. Stryer Fig. 4.18 Ion-exchange chromatography

40. Stryer p. 92 Agents for N-terminal analysis

41. Stryer Fig. 4.20

42. Stryer Fig. 4.21

43. Animation: Edman Degradation http://www.wiley.com/college/fob/quiz/quiz05/f5-15.html

44. Stryer Fig. 4.22 Separation of PTH-aa by HPLC.

45. 2. Partially digest intact protein with specific agents.

46. Stryer Fig. 4.23

47. Stryer Fig. 4.24

49. 3. Sequence the fragments and align overlapping sequences.

50. Stryer Fig. 4.25