powerpoint presentation by kru's biology youtube channel.

1. Adopted from Nilsen and cox – Lehninger principles of biochemistry (sixth edition)
Chapter 3 : Amino Acids, Peptides and
Proteins

2. Learning Objectives
• To know the structure and naming of all 20 protein amino acids
• To know the structure and properties of peptides and the particularly
the structure of the peptide bond.
• Ionization behavior of amino acids and peptides at different pH’s.
• To know the general pKa’s of amino acids: their carboxyl's, aminos, the
R-group weak acids.

3. Some Function of proteins

4. Amino Acids:
Building Blocks of Protein
• Proteins are linear heteropolymers of α-amino acids
• Amino acids have properties that are well-suited to carry
out a variety of biological functions
– Capacity to polymerize
– Useful acid-base properties
– Varied physical properties
– Varied chemical functionality

5. Amino acids share many features, differing
only at the R substituent

6. L and D forms

7. Carbon Numbering System
A
the carboxyl carbon of an amino acid would be C-1 and the
alpha carbon would be C-2.

8. Amino Acids: Classification
Common amino acids can be placed in five basic
groups depending on their R substituents:
• Nonpolar, aliphatic R groups (7)
• Aromatic R groups (3)
• Polar, uncharged R groups (5)
• Positively charged R groups (3)
• Negatively charged R groups (2)

9. Invented the One Letter
Amino Acid Code.

15. Spectrophotometry

16. UV light Absorption by Proteins – due to 2 Amino Acids

17. Cysteine can form Disulfide Bonds

18. Uncommon Amino Acids
Extra functional groups added by
modification reactions are shown in red.
The four carbon backbones are shaded in
yellow

19. Amino acids in Proteins Can be Reversibly Modified
A
• Reversible amino acid modifications
involved in regulation of protein activity.
• Phosphorylation is the most common type
of regulatory modification.

20. Non Protein Amino Acids

21. Toxic Amino Acids
A search for compounds producing
Yunnan Sudden Unexplained Deaths
found related to eating a mushroom.
Trogia venenata Zhu LHalford, B. C+E News Feb 13, 2012

22. Which Form Occurs in Water ?
A zwitterion can
act as either an
acid (proton
donor)
A zwitterion can
act a base
(proton
acceptor)

23. Glycine Acid/Base Titration

24. Compare Amino Acids to Simple Carboxylic Acids and
Amines

25. Glutamate has 3 pKa’s

26. Histidine has 3 pKa’s

27. How to Calculate the PI When the Side Chain
is Ionizable
• Identify species that carries a net zero charge
• Identify pKa value that defines the acid strength of this
zwitterion: (pK2)
• Identify pKa value that defines the base strength of
this zwitterion: (pK1)
• Take the average of these two pKa values
What is the pI of histidine?

28. Peptide Bond Formation by condensation
Where does this occur? Where does this occur?

29. Structure of a Simple Peptide
Ser-Gly-Tyr-Ala-Leu or SGYAL
Peptide name (placed left)beginning with the amino-terminal residue
The peptide bonds are shaded in yellow; the R groups are in red.

30. Naming peptides:
start at the N-terminus
• Using full amino acid names
– Serylglycyltyrosylalanylleucine
• Using the three-letter code abbreviation
– Ser-Gly-Tyr-Ala-Leu
• For longer peptides (like proteins) the one-
letter code can be used
– SGYAL

31. AEGK

32. Aspartame
A

33. Peptides: A Variety of Functions
• Hormones and pheromones
– insulin (think sugar)
– oxytocin (think childbirth)
– sex-peptide (think fruit fly mating)
• Neuropeptides
– substance P (pain mediator)
• Antibiotics
– polymyxin B (for Gram – bacteria)
– bacitracin (for Gram + bacteria)
• Protection, e.g., toxins
– amanitin (mushrooms)
– conotoxin (cone snails)
– chlorotoxin (scorpions)

34. Proteins are:
• Polypeptides (covalently linked α-amino acids) + possibly:
●
cofactors
• functional non-amino acid component
• metal ions or organic molecules
●
coenzymes
• organic cofactors
• NAD+ in lactate dehydrogenase
●
prosthetic groups
●
covalently attached cofactors
●
heme in myoglobin
●
other modifications

38. Things to Know
1. Know Structure and chemistry of all 20 amino acids.
2. Approximate pKa of amino acid ionizable groups and
their ionization state at different pH’s.
3. Modifications of amino acids in proteins.
4. Disulfide bonds, make and break them, and diagram
them.
5. The Peptide bond, make and break it, and diagram them.
6. EOC Problems 1, 2, 3a, 4-7: we will have problems to
solve (clicker questions) in class like these. Please
practice these well before class.

39. Protein Purification

40. Learning Objectives
1. Know how each classical method of protein purification
works.
2. Know how to measure protein and then calculate total
activity and specific activity in each protein purification
step.
3. Know how to evaluate protein purity.
4. Know how 2D PAGE gels work.
5. Know how molecular methods of protein purification work.

41. A mixture of proteins can be separated
• Separation relies on differences in physical and
chemical properties
– Charge
– Size
– Affinity for a ligand
– Solubility
– Hydrophobicity
– Thermal stability
• Chromatography is commonly used for
preparative separation

42. Solubility of Some Proteins in Ammonium Sulfate

43. Classical Protein Purification Methods
The Oldest: Ammonium Sulfate Fractionation
Clear Cell
Free Extract
Cloudy
Clear
Supernate

44. Ammonium Sulfate Fractionation Table

45. Ammonium Sulfate Fractionation
Example Results: 0 – 20% ppt, 20-40% ppt, 40 -60%
ppt, 60-80% ppt and 80-100% ppt….where is your
protein?

46. These Fractions Need to be Assayed
For:
1. Total Protein can be done by
a. Absorbance at 280 nm
b. Colorimetric tests for protein
Lowry Assay, Bradford Assay
2. Your Specific Protein
a. specific enzyme assay, or
b. specific binding assay, or
c. unique spectral property.

47. Example Results – each Cut in 100 ml of Buffer
(NH4)2SO4 Cut Total Protein Your Enzyme
Crude Extract 1.73 g 3,895 mM/sec
0 – 20% 452 mg 0.1 mM/sec
20 – 40% 323 mg 3,560 mM/sec
40 – 60% 541 mg 12 mM/sec
60 – 80% 329 mg 0.1 mM/sec
80 – 100% 78 mg 0.01 mM/sec
Total Enzyme Activity Specific Activity
Crude: 3.9 x 103 mM/sec 2.25 (mM/sec)/mg protein
20-40 Cut 3.56 x 103 mM/sec 11.0 (mM/sec)/mg protein
(91% orig. activity) 4.9X fold purified

48. Example Results – each Cut in 100 ml of Buffer
(NH4)2SO4 Cut Total Protein Your Enzyme
Cell Free Extract1.73 g 3,895 mM/sec
0 – 20% 452 mg 0.1 mM/sec
20 – 40% 323 mg 3,560 mM/sec
40 – 60% 541 mg 12 mM/sec
60 – 80% 329 mg 0.1 mM/sec
80 – 100% 78 mg 0.01 mM/sec
How did you get rid of the high conc of
(NH4)2SO4 ?

49. Dialysis – Getting Rid of (NH4)2SO4 or Change Buffers

50. Column Chromatography Principle

51. Columns Connected to Fraction Collectors
Tube from column
attached here

52. Separation by Charge: Ion Exchange

53. Ion Exchangers

54. Ion Exchange Chromatography

55. Gel Filtration or Size Exclusion Chromatography

56. Separation by Size: Gel or Size Exclusion

57. Gel Filtration Media

58. Gel Filtration or Exclusion Chromatography

59. Gel Filtration Gives an Idea of Molecular Weight

60. Separation by Binding Affinity

61. Units = μM/sec or mM/min = rate…Enzymes convert S  P
Fold Purification = (Specific Activity at Step) / (Sp. Act. Crude Extract)
Final Purification is 1,500 fold pure.
Purification Table

62. 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

63. Polyacrylamide Gel Electrophoresis

64. Gel Showing
Steps in
Purification
Purification of RecA
from E. coli.

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

66. SDS-PAGE can be used to calculate the molecular
weight of a protein

67. Isoelectric focusing can be used to determine the pI
of a protein

68. A

69. 2-D Gels Start with Isoelectric Focusing

70. 2-D Gels End with SDS PAGE

71. Isoelectric focusing and SDS-PAGE are combined in
2D electrophoresis
A 2-D Gel of Escherichia coli Cytoplasm

72. Molecular Methods of Protein Purification
1.Isolate the gene…restriction enzymes, separation DNA on
agarose gels, insert gene into a plasmid behind an active
promoter (turns on gene), and with a “tag” (such has
six-histidines) or Maltose Binding Protein or other
tag. The tag makes this a fusion protein:
Protein-his-his-his-his-his-his or Protein-MBP
2.Insert the plasmid into a bacterium (usually E. coli) and turn-
on the promoter to express the fusion-protein in large
quantities (the protein can be 10-30% cell volume!).
3.Lyse cells, fusion-protein binds affinity column which
after binding and washing provides the fusion protein
essentially pure.
4.Cleave off the his tag, dialyze  pure protein.

73. Molecular Methods of Protein
Purification
New England BioLabs, Inc

74. Expression Purification
Time after induction (hours)
0 0.5 1.0 1.5 2.0 2.5 3.0
Production and Purification of Tescalcin
Totallysate
Unbound
Wash2
Wash3
Wash1
Elute2
Elute1
115
93
50
36
29
21
KDa
His6-Tsc
His6-Tsc
cDNA cloned in pET15b expression vector
E. coli (BL21) transformation
Induction with IPTG
Mechanical lysis
Ni-NTA metal affinity chomatography
Elution with imidazol
by Erasmo Perera, FIU student

75. Things to Know and Do Before Class
1. Know each method used Purify Proteins and How they
Work.
2. Calculation of Total protein and Specific Activity in the
steps of protein purification.
3. Testing for protein purity.
4. How to do 2D PAGE gels.
5. Molecular Methods of Protein Purification.
6. Be able to do EOC Problems: 8-11, 13, 15 (protein
purification), 16.