1. Pratt & Cornely Chapter 4
Amino Acids
Pratt & Cornely
Chapter 4

2. Amino Acid Structure
Alpha carbon
Sidechain
Proteins
peptides

3. Stereochemisty L-amino acids racemization Glycine R/S vs D/L
L-isoleucine
racemization

4. Common Amino Acids

5. Which amino acid(s)... Is achiral? Has a secondary amino group?
Has a chiral sidechain?
Form these derivatives:

6. Acid/base chemistry Charged amino acids
Other amino acids are ionizable

7. Ionization of Amino Acids
Polyprotic acids
Alanine
pKa1 = 2.4
pKa2 = 9.9
Zwitterion
Isoelectric point
More acidic
than typical
carboxylic acid

8. Titration Curve

9. Titration Curve
Which amino acid?

10. Ionization States Of Amino AcidsG F E D C B A

11. Disulfide bond formation

12. Peptide bonds Amide bond Primary structure N- and C-terminus
Condensation and hydrolysis

13. Polypeptides

14. Drawing Peptides Sidechains Stereochemistry Ionization states
Example: Draw the peptide AHSCVE at pH 8.
Steps
Backbone
Stereochemistry
Sidechains
Check ionization

15. Example: Draw the peptide AHSCVE at pH 8.

17. Basis of secondary Structure
Polarity
Rigidity
Cis/trans

18. Conformational Constraint
NOT cis/trans

19. Dihedral Angles

20. Ramachandran Plots

21. Ramachandran Plots

22. Alpha Helix
Right handed
Polarity
n and n + 4
Gly and Pro

23. Helical Wheel
Problem 31. The sequence of a domain of the gp160 protein(HIV) is shown below using one-letter codes for the amino acids. Plot this sequence on the helical wheel. What do you notice about the amino acid residues on either side of the wheel? MRVKEKYQHLWRWGWRWG

24. Helical Wheel
The sequence of a domain of the gp160 protein(HIV) is shown below using one-letter codes for the amino acids. Plot this sequence on the helical wheel. What do you notice about the amino acid residues on either side of the wheel? MRVKEKYQHLWRWGWRWG M R Q W E K R W H G R W Y G K W V L

25. Beta Sheets
Alternating sidechains can lead to amphipathic sheets

27. Irregular Secondary Structure
Nonrepeating loops and turns
Change of direction
Turns have about 4 residues
Internal H-bonds
Gly, Pro

28. Tertiary Structure Too many shapes to memorize
But not an infinite number of possibilities
Take away the ability to read a paper
Discussions of motifs and why important
Discussion of domains and why important

29. Motifs (Super Secondary Structure)
Recognizable combinations of helices, loops, and sheets
Match
Helix-loop-helix
Helix bundle
Hairpin
b-sandwich

30. Studying Motifs Some Motifs are highly studied Know the lingo
Leucine zipper
Zinc finger
Often have recurring applications

31. Domains
Discrete, independently folded unit (may maintain shape when cleaved on loop)
May have separate activities: “ATP binding domain” or “catalytic domain”
Similar activity = similar structure across many proteins
Binding pockets at interfaces
How many domains?

32. Common Domains

33. Major classes of globular proteins
Mostly a
Mostly b
a/b combination
Little secondary

34. Thermodynamics of protein folding
DG might be 40 kJ/mol for small protein (about 2 H-bonds)
Hydrophobic effect is important...but the most important?

35. Thermodynamic Contributions to RNase
Pace, C.N. Meth. Enz. 1995, 259,

36. Other Stabilization
Ion pair (salt bridge)
Zinc finger
Disulfide bond

37. Protein Folding Denature/renature Native structure
Domains fold separately
Global vs. local minima
Molecular chaperones
Problem 40. Proteins can be denatured by agents that alter the balance of weak noncovalent forces that maintain the native conformation. How would these agents cause a protein to denature? Heat, pH, detergent, 2-mercaptoethanol

38. Protein processing

39. Thermodynamics and Kinetics
Levinthal’s paradox: not random sampling of all possible conformations
Energy funnel
Series of irreversible steps
Entropy traps

40. Quaternary Structure Multiple subunits: Oligomers
Homodimer, heterotrimer
Advantages
Economy
Stability
regulation a3
a2b2

41. Protein Purification Chromatography Electrophoresis Affinity HPLC
Size Exclusion (gel filtration)
Ion exchange
Electrophoresis
Isoelectric focusing
SDS-PAGE

42. Size exclusion Chromatography

43. Ion Exchange Chromatography

44. Isoelectric Focusing
From wikipedia

45. SDS PAGE Sodium dodecyl sulfate Polyacrylamide gel electrophoresis
Analaysis technique
Small proteins move faster

46. Amino Acid Sequencing Purify protein Break disulfide bonds
Enzymatic digest (protease)
Edman degradation
Overlap fragments

47. Partial Digestion

48. Partial Digestion

49. Edman Degradation

50. Sequencing Oligopeptides

51. X-Ray crystallography