This chapter discusses amino acids, peptides, and proteins. It covers the structure and properties of amino acids, how they combine to form peptides and proteins, and the various functions of proteins, including catalysis, transport, structure, and motion. Methods for analyzing, separating, and purifying proteins are also described, such as ion exchange chromatography, size exclusion chromatography, electrophoresis, and protein sequencing. Finally, the chapter classifies common amino acids based on the properties of their variable side chains.

1. CHAPTER 3
Amino Acids, Peptides,
Proteins
• Structure and naming of amino acids
• Structure and properties of peptides
• Ionization behavior of amino acids and peptides
• Purification and assay methods
• Peptide sequencing and chemical synthesis
• Protein sequence analysis

2. Proteins: Main Agents of
Biological Function
• Catalysis:
–enolase (in the glycolytic pathway)
–DNA polymerase (in DNA replication)
• Transport:
–hemoglobin (transports O2 in the blood)
–lactose permease (transports lactose across the cell membrane)
• Structure:
–collagen (connective tissue)
–keratin (hair, nails, feathers, horns)
• Motion:
–myosin (muscle tissue)
–actin (muscle tissue, cell motility)

3. Amino Acids: Building Blocks of
Protein
• Proteins are 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

4. Amino Acids: Atom Naming
• Organic nomenclature: start from one end
• Biochemical designation: start from
α-carbon and go down the R-group

5. Most α-Amino Acids are Chiral
• The α-carbon has always
four substituents and is
tetrahedral
• All (except proline) have an
acidic carboxyl group, a
basic amino group, and an
alpha hydrogen connected
to the α-carbon
• Each amino acid has an
unique fourth substituent R
• In glycine, the fourth
substituent is also hydrogen

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

7. Aliphatic Amino Acids
• http://en.wikipedia.org/wiki/File:Aa.svg

8. Aromatic Amino Acids
• http://en.wikipedia.org/wiki/File:Aa.svg

9. Charged Amino Acids
• http://en.wikipedia.org/wiki/File:Aa.svg

10. Polar Amino Acids
• http://en.wikipedia.org/wiki/File:Aa.svg

11. Special Amino Acids
• http://en.wikipedia.org/wiki/File:Aa.svg

13. Not incorporated by ribosomes
Arise by post-translational modifications of
proteins
Reversible modifications, esp.
phosphorylation is important in regulation
and signaling
Uncommon Amino
Acids in Proteins

14. The Genetic Code is organized
by Amino Acid Properties

15. Ionization
At acidic pH, the carboxyl
group is protonated
and the amino acid is
in the cationic form
At neutral pH, the
carboxyl group is
deprotonated but the
amino group is
protonated. The net
charge is zero; such
ions are called
Zwitterions
At alkaline pH, the amino
group is neutral –NH2
and the amino acid is
in the anionic form.

16. Substituent effects on pKa Values
α-carboxy group is much more acidic than in carboxylic acids
α-amino group is slightly less basic than in amines

17. Amino Acids Can
Act as Buffers
Amino acids with
uncharged side-chains,
such as glycine, have two
pKa values:
The pKa of the α-carboxyl
group is 2.34
The pKa of the α-amino
group is 9.6
It can act as a buffer in
two pH regimes.

18. Amino Acids Carry a Net Charge
of Zero at a Specific pH
•Zwitterions predominate at pH values between the pKa values
of amino and carboxyl group
•For amino acid without ionizable side chains, the Isoelectric
Point (equivalence point, pI) is
• At this point, the net charge is zero
– AA is least soluble in water
– AA does not migrate in electric field
2
21 pKpK
pI
+
=

19. Ionizable Side Chains Can Show
Up in Titration Curves
• Ionizable side chains
can be also titrated
• Titration curves are
now more complex
• pKa values are
discernable if two pKa
values are more than
two pH units apart
Why is the side-chain
pK so much higher?

20. 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: (pKR)
• Take the average of these
two pKa values

21. Peptides and Peptide bonds
Peptide bond in
a di-peptide
“Peptides” are
small
condensation
products of
amino acids
They are “small”
compared to
proteins (di, tri,
tetra… oligo-)

22. Peptide Ends are Not the Same
Numbering starts from the amino terminus
AA1 AA2 AA3 AA4 AA5

23. 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

24. 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)

25. Proteins are:
• Cofactor is a general term for functional non-amino acid component
– Metal ions or organic molecules
• Coenzyme is used to designate an organic cofactors
– NAD+
in lactate dehydrogenase
• Prosthetic groups are covalently attached cofactors
– Heme in myoglobin
• Polypeptides (covalently linked α-amino acids) + possibly –
• cofactors,
• coenzymes,
• prosthetic groups,
• other modifications

26. Polypeptide Size in Some Proteins

27. Classes of Conjugated Proteins

28. Peptides and Proteins-
Burning Questions
Sequence and composition?
Three-dimensional structure?
Folding Mechanism?
Biochemical role?
Functional regulation?
Molecular interactions with small and macro-molecules?
Structural and sequence relatives?
Cellular and sub-cellular localization?
Physical and chemical properties?

29. Purification – Fractionation of
Protein Mixtures
• Separation relies on differences in physico-
chemical properties
– Solubility – Selective Precipitation (Centrifugation)
– Thermal stability --
– Charge --Electrophoresis, Isoelectric Focusing, IEC
– Size – Dialysis, Sedimentation (Centrifugation), GFC
– Affinity for a ligand – “Pull down” assays (Centrifugation),
AC
– Hydrophobicity (HIC)
• Chromatography is commonly used for
preparative separation

30. http://www.salinesystems.org/content/figures/1746-1448-4-1-2-l.jpg
Protein Fractionation

31. Separation by Charge
•Ion Exchange Chromatography
•Anion exchange
Matrix positive
Proteins negative
Displaced by anions
•Cation exchange – Opposite
• pH determines net charge on
Proteins
•Salt concentration gradient
•Native gel electrophoresis
•Iso-electric Focusing

32. Separation by
Size
• Size exclusion (Gel
Filtration)
Chromatography
– Loading vol. <5% of
column volume
– Samples diluted
• Dialysis or Centrifugal
concentrators

33. Separation by
Affinity
• Affinity
Chromatography
• Free Ligand-Beads --
centrifugation
• Ligand-Magnetic-
Beads
• Immuno-assays on
solid supports

34. 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

35. SDS PAGE: Molecular Weight
• SDS – sodium dodecyl
sulfate – a detergent
• SDS micelles binds 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
-

36. Protein
Sequencing

37. Spectroscopic Detection of Aromatic
Amino Acids
• The aromatic amino acids
absorb light in the UV
region
• Proteins typically have
UV absorbance maxima
around 275-280 nm
• Tryptophan and tyrosine
are the strongest
chromophores
• Concentration can be
determined by UV-visible
spectrophotometry using
Beers law: A = ε·c·l

38. Chapter 3: Summary
In this chapter, we learned about:
• The many biological functions of peptides and
proteins
• The structures and names of amino acids found in
proteins
• The ionization properties of amino acids and
peptides
• The methods for separation and analysis of
proteins

39. Nonpolar, Aliphatic R Groups

40. Aromatic R Groups
Also
Hydrophobic
These amino
acid side
chains absorb
UV light at
270-280 nm

41. Polar,
Uncharged R
Groups
These amino
acids side
chains can
form
hydrogen
bonding
Cysteine can
form disulfide
bonds

42. Basic R
Groups

43. Acidic R Groups