1. Protein Building Blocks: Amino Acids, Peptides and Polypeptides

2. Protein Functions: Enzymes – catalysis Tissue structure Cell structure
Hormones
Hormone receptors
Ion channels
Transporters
Transcription factors
Contraction
--etc.

3. Protein Synthesis nucleus transcription transcription translation DNA
genes
mRNA
(codon sequence for amino acids)
Protein
(specific amino acid sequence)
transcription
transcription
translation
WHS

4. “Proteome”: a catalog of all proteins
How Many Proteins?
“Proteome”: a catalog of all proteins
Life Technologies

5. C is a chiral center (4 different substituents).
Amino Acids R
H2N C COOH H α R
H3N C COO H + _ α
Zwitterion
(at physiological pH) R
H3N C COO H + _ α
C is a chiral center (4 different substituents).
Optical isomers:
In Proteins
L – amino acid
D – amino acid

6. Hydrophobic Hydrophilic R-groups in Amino Acids Non-polar; hydrocarbon
Doesn’t interact with H2O
Polar/charged
Interacts with H2O; hydrogen bonds to N, O
In General:
Hydrophobic amino acids are in the interior of a folded protein.
Hydrophilic amino acids are on the surface in contact with water.

7. Common Amino Acids (20)

8. Common Amino Acids (cont’d))

10. Peptides and the Peptide Bond
H3N C COO + _ α
CH3
glycine
alanine
( H2O)
Peptide bond
Glycylalanine
(a dipeptide)

11. Methionine Enkephalin
Polypeptides
Methionine Enkephalin
a pentapeptide (five amino acids)
Tyr-Gly-Gly-Phe-Met
H3N-Tyr-Gly-Gly-Phe-Met-COO + or
H-Tyr-Gly-Gly-Phe-Met-OH or
Amino terminus;
NH2-terminus
N-terminus
Carboxyl terminus;
COOH-terminus
C-terminus
(An amino acid in a peptide is often called a “residue”)

12. A Derived Amino Acid--Cystine
Oxidation of two cysteine side chain thiol groups
(a disulfide bond)

13. Antidiuretic Hormone—Vasopressin
(a nonapeptide)
H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly(NH2)
S S
glycinamide O
C NH2

14. Insulin

15. Peptide or Protein? Peptide: <50 amino acids
Protein: >50 amino acids
Median protein size: ≈ 400 amino acids
Largest protein (titin): ≈ 30,000 amino acids

16. Acid dissociation equilibrium constant Kα :
Acid - Base Review
Definitions:
Acid: proton donor
Base: proton acceptor
Acid dissociation equilibrium constant Kα : Ka
HA ↔ H A‾ +
conjugate conjugate
acid base
The concentrations ( [ ] ) at equilibrium:
[H ] [A ]
[HA] + _ Ka =

17. + _
log Ka = log +
log Ka = log [H ] log
[A ]
[HA] _ +
log [H ] = log [Ka] log
[A ]
[HA] _
pH pKa
[A ]
[HA]
pH = pKa log _
Henderson-Hasselbalch Equation

18. [A ]
[HA]
pH pKa = log _
[A ]
[HA]
pH pKa _

19. pKa Values of Some Amino Acid Side Chains
(low pKa, acidic; high pKa basic)

20. Amino Acid Titration: Alanine
NaOH added 10 8 6 4 2 pH

21. (The pI is half way between the pKa’s that border the zwitterion.)
Aspartic acid
Lysine
Isoelectric point ( pI ): pH where equal number of + and – charges (zwitterion).
(The pI is half way between the pKa’s that border the zwitterion.)

22. Proteins also have a pI
pI (isoelectric point)
Equal + and – charges (no net charge)
Doesn’t migrate in an electric field
Depends on the amino acid composition
Human serum albumin (585 amino acids)
36 Aspartates
61 Glutamates
57 Lysines
24 Arginines
16 Histidines
pI = 5.9 pI
+ charged
charged _

23. Clinical Use of Protein Electrophoresis
Agarose gel electrophoresis of plasma proteins
carried out at pH 8.6

24. e.g., multiple myeloma

25. Separation of a mixture of proteins by pI:
The Proteome: 2-Dimensional Electrophoresis
The method:
Separation of a mixture of proteins by pI:
Isoelectric focusing
pH gradient + _
Sodium dodecyl sulfate gel electrophoresis
(separation of proteins by size) + _
Then stain gel for protein

28. Digest protein with an enzyme: trypsin (cleaves after a Lys or Arg)
Cut out spot
Digest protein with an enzyme:
trypsin (cleaves after a Lys or Arg)
Separate fragments by HPLC*, measure mass
*High Performance Liquid
Chromatography
Mass spectrometer
Peptide
mass
Tandem mass spectrometer
Peptide
sequence

29. Cancer Proteomics:
Determine protein/peptide biomarkers
Early Diagnosis
Staging (severity)
Molecular-targeted therapy
Monitoring therapy