Download Amino Acids and the Primary Structure of Proteins

Amino Acids and the
Primary Structure of Proteins
Important biological functions of proteins
1. Enzymes, the biochemical catalysts
2. Storage and transport of biochemical molecules
3. Physical cell support and shape (tubulin, actin,
collagen)
4. Mechanical movement (flagella, mitosis,
muscles)
(continued)
Amino Acids and the
Primary Structure of Proteins
5. Decoding cell information (translation,
regulation of gene expression)
6. Hormones or hormone receptors (regulation of
cellular processes)
7. Other specialized functions (antibodies, toxins
etc)
Zwitterionic form of amino
acids
• Under normal cellular conditions amino
acids are zwitterions (dipolar ions):
Amino group =
Carboxyl group =
-NH3+
-COO-
Two representations of an amino
acid at neutral pH
Titration Curve for
Alanine
• Titration curves
are used to
determine pKa
values
• pK1 = 2.4
• pK2 = 9.9
• pIAla = isoelectric
point
Aliphatic R Groups
• Glycine (Gly, G) - the a-carbon is not chiral
since there are two H’s attached (R=H)
• Four amino acids have saturated side chains:
Alanine (Ala, A) Valine (Val, V)
Leucine (Leu, L) Isoleucine (Ile, I)
• Proline (Pro, P) 3-carbon chain connects
a-C and N
Stereoisomers of Isoleucine
• Ile has 2 chiral carbons, 4 possible stereoisomers
Aromatic Amino Acids
Methionine and Cysteine
Formation of Cystine
Histidine, Lysine, and Arginine
Aspartate, Glutamate
Asparagine, Glutamine
Peptide Chain Nomenclature
• Amino acid “residues” compose peptide chains
• Peptide chains are numbered from the N (amino)
terminus to the C (carboxyl) terminus
• Example: (N) Gly-Arg-Phe-Ala-Lys (C)
(or GRFAK)
• Formation of peptide bonds eliminates the
ionizable a-carboxyl and a-amino groups of the
free amino acids
Peptide Sequencing
Edmann Degradation
O
H2N
N C S
R1
H
O
O
H
R1
NH 2
N C S
H
H
O
R2
N
H
H
N
O
O
R2
N
H
H
N
O
O
R3
R4
N
H
O
R4
O
R3
N
H
O
R1
HN
N C S
H
H
R2
N
H
H
N
O
O
R3
R4
N
H
O
Edmann Degradation (cont.)
O
R1
R2
N
H
HN
H
N
O
O
R4
N
H
R3
O
N C S
H
CF3COOH
H
O
H
O
R1
R2
S
N
H
+
H
N
H3N
O
N C6H5
H
O
R3
R4
N
H
O
Edmann Degradation (cont.)
H
O
H
O
R1
S
N
H
N C6H5
H
O
O
OH
R1
OH
R1
S
N
H
N C6H5
H
N
N
H
H
S
C6H5
Edmann Degradation (cont.)
O
O
OH
R1
OH
R1
S
N
H
H
N
N
H
C6H5
S
N C6H5
H
O
R1
N
N
H
S
C6H5
Cleaving Disulfide bonds and
Protecting the thiols formed
• Disulfide bonds in proteins must be cleaved:
(1) To permit isolation of the PTH-cysteine
during the Edman procedure
(2) To separate peptide chains
• Treatment with thiol compounds reduces the
(R-S-S-R) cystine bond to two cysteine
(R-SH) residues
• Thiols are protected with iodoacetate
Further Protein Sequencing
Strategies
• Proteins may be too large to be sequenced
completely by the Edman method
• Proteases (enzymes cleaving peptide bonds)
and chemical agents are used to selectively
cleave the protein into smaller fragments
• Cyanogen bromide (BrCN) cleaves
polypeptides at the C-terminus of Met residues
Protease Enzymes
cleave specific peptide bonds
• Chymotrypsin - carbonyl side of aromatic or
bulky noncharged aliphatic residues (e.g. Phe,
Tyr, Trp, Leu)
• Trypsin - carbonyl side, basic residues (Lys,Arg).
• Staphylococcus aureus V8 protease - carbonyl
side of negatively charged residues (Glu, Asp).
NOTE: in 50mM ammonium bicarbonate cleaves
only at Glu.