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蛋白質體學
Proteomics 2011
Amino acids,peptides
and proteins
陳威戎
2011. 09. 19
Outline
1. Amino Acids
2. Peptides and Proteins
3. Covalent Structure of Proteins
Amino Acids
Proteins are polymers of amino acids, with each
amino acid residues joined to its neighbor by a
specific covalent bond.
Twenty different amino acids are commonly found in
proteins.
First: asparagine (1806) ; last: threonine (1938).
Names derived from the sources:
Asparagine – asparagus
Glutamate – wheat gluten
Tyrosine – cheese
Glycine – sweet taste
Amino acids share common structural features
Two conventions used to identify the carbons in
an amino acid
The α-carbon atom is a chiral center.
L-Form Amino Acid Structure
Carboxylic group
Amino group
+
H3 N
R group
COO
a
-
H
H = Glycine
CH3 = Alanine
Juang RH (2004) BCbasics
Northwest line Chung-San line
-C-C-C-N-C-N
=
Aromatic
N+
Trp W
Arg R
-C-
Basic
-OH
Tyr Y
Lys K
+
-CN
Nan-Kan line
-C-CONH2
-C-C-CONH2
Asn N
Gln Q Amide
Asp D
Glu E Acidic
-C-COOH
-C-C-COOH
-C-
-C-C C
N N+
His H
Central line Gly G
Phe F
Ala A
A
Val V
Ile I
-CH3
C C
-C
C
-C-C-C
-H
-C-OH
Ser S
Cys C
-C-SH
Circular line
South line
Non-polar
Polar
-C-C
OH
Thr T
Met M
Hydroxy
Sulfur
-C-C-S-C
Aliphatic
Leu L
C
-C-C-C
C
C
C
HN C-COOH
a
Pro P
Imino,
Circular
Juang RH (2004) BCbasics
-C-C-C-C-NH3
Amino Acid Subway Map
Absorption of ultraviolet light by aromatic amino acids
Reversible formation of the disulfide bond
Classification of Amino Acids by Polarity
NONPOLAR
POLAR
Acidic
Neutral
Basic
Asp
Asn Ser
Arg
Cys
Tyr
His
Gln
Thr
Lys
Glu
Gly
Ala Ile
Phe Trp
Val Leu Met Pro
Polar or non-polar, it is the bases of the amino acid properties.
Juang RH (2003) Biochemistry
Uncommon amino acids also have important functions
Uncommon amino acids also have important functions
Hydride, Hydrogen and Proton
hydride
1s
-
-
Proton
+
+
-
-
+
-
Hydrogen atom
1
H
1.008
Juang RH (2004) BCbasics
Proton Is Adsorbed or Desorbed
Proton： abundant and small, affects the charge of a molecule
lone pair
electrons
Amino
High pKa Low
N H
H+
H+
N H
H
H
Low pKa High
Carboxylic
C
O H
O
O
C
O
H+
Ampholyte contains both positive and negative groups on its molecule
Juang RH (2004) BCbasics
Amino acids can act as acids and bases
Acidic environment
Neutral environment
Alkaline environment
pK2 ~ 9
NH2 H+
R-C-H
COOH
NH2 H+
R-C-H
COOpK1 ~ 2
+1
NH2
R-C-H
COO-
5.5
0
Isoelectric point
-1
Juang RH (2004) BCbasics
Amino Acids Have Buffering Effect
pH 12
★
pK2
Isoelectric point =
pI
9
NH2 H+
6
H-C-R
COO-
3
★
pK1 + pK2
2
pK1
0
[OH] →
Juang RH (2004) BCbasics
Environment pH vs Protein Charge
Buffer pH
10
9
8
7
Isoelectric point,
pI
+
6
5
4
3
0
-
-
Net Charge of a Protein
Juang RH (2004) BCbasics
pKa of Amino Acid Residues
Residues on amino acids can release or accept protons
a -COOH
R -COOH
His -Imidazole·H+
Cys -SH
Tyr -OH
a -NH3+
R -NH3+
a -COOR -COO-
+ H+ pKa = 1.8~2.4
+ H+ pKa = 3.9~4.3
His -Imidazole + H+ pKa = 6.0
+ H+ pKa = 8.3
Cys -S+ H+ pKa = 10
Tyr -Oa -NH2
R -NH2
+ H+ pKa = 8.8~11
+ H+ pKa = 10~12.5
Smaller pKa releases proton easier
Only His has the residue with a neutral pKa (imidazole)
pKa of a carboxylic or amino groups is lower than pKa of the R residues
Juang RH (2004) BCbasics
pKa of Amino Acids
Amino acids
-COOH -NH2
Gly
Ala
Val
Leu
Ile
Ser
Thr
Met
Phe
Trp
Asn
Gln
Pro
Asp
Glu
His
Cys
Tyr
Lys
Arg
2.34
2.34
2.32
2.36
2.36
2.21
2.63
2.28
1.83
2.38
2.02
2.17
1.99
2.09
2.19
1.82
1.71
2.20
2.18
2.17
G
A
V
L
I
S
T
M
F
W
N
Q
P
D
E
H
C
Y
K
R
9.60
9.69
9.62
9.68
9.68
9.15
10.4
9.21
9.13
9.39
8.80
9.13
10.6
9.82
9.67
9.17
10.8
9.11
8.95
9.04
-R
pH
two pKa
pK2
pI
pK1
pK1 + pK2
2
three pKa
pK3
3.86
4.25
6.0
8.33
10.07
10.53
12.48
pK2
pK1
?
?
pI ?
[OH-]
Juang RH (2004) BCbasics
H
first
HOOC-CH2-C-COOH
NH3+
+1
pK1 = 2.1
H
second
HOOC-CH2-C-COO-
Aspartic acid
Isoelectric point is the average
of the two pKa flanking the
zero net-charged form
2.1 + 3.9
= 3.0
2
Isoelectric point
0
NH3+
pK2 = 3.9
H
-OOC-CH -C-COO2
-1
NH3+ third
pK3 = 9.8
H
-OOC-CH -C-COO2
NH2
-2
-2
pK3
-1
pK2
pK1
0
+1
[OH]
Juang RH (2004) BCbasics
Titration curves predict the charge of amino acids
Isoelectric point,
Isoelectric pH, pI
Amino acids differ in their acid-base properties
Amino acids differ in their acid-base properties
Quiz 1: Net electric charge and pI of a peptide
A peptide has the sequence :
Glu-His-Trp-Ser-Gly-Leu-Arg-Pro-Gly
1. What is the net charge of this peptide at pH 3, 8,
and 11?
2. Estimate the pI for this peptide.
Peptides are chains of amino acids
Formation of a peptide bond by condensation
Peptides are chains of amino acids
The pentapeptide serylglycyltyrosylalanylleucine, or
Ser-Gly-Tyr-Ala-Leu
Biologically active peptides and polypeptides occur in a
vast range of sizes
Peptides have characteristic
amino acid compositions
Some proteins contain chemical groups other than
amino acids
There are several levels of protein structure
Proteins can be separated and
purified
Crude extract
Fractionation
Ammonium sulfate
(salting out)
Dialysis
Column
chromatography
Proteins can be separated and characterized by
electrophoresis
The amino acid sequences of
millions of proteins have been
determined
Frederick Sanger
Short polypeptides are sequenced using
automated procedures
Large proteins must be sequenced in
smaller segments
1. Breaking disulfide bonds
2. Cleaving the polypeptide chain: proteases
3. Sequencing of peptides
4. Ordering peptide fragments
5. Locating disulfide bonds
Breaking disulfide bonds in proteins
Cleaving the polypeptide
chain: proteases
Cleaving proteins and sequencing and
ordering the peptide fragments
Amino acid sequences can also be
deduced by other methods
1. New methods based on mass spectrometry permit
sequencing of short polypeptides (20-30 a.a.) in just a
few minutes.
2. Development of rapid DNA sequencing methods.
Quiz 2: Sequence determination of a nonapeptide
1. A nonapeptide was determined to have the following amino acid
composition: (Lys)2, (Gly)2, (Phe)2, His, Leu, Met.
2. The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene
(FDNB) and then hydrolyzed; 2,4-dinitrophenylhistidine was identified
by HPLC.
3. When the native peptide was exposed to cyanogen bromide (CNBr), an
octapeptide and free glycine were recovered.
4. Incubation of the native peptide with trypsin gave a pentapeptide, a
tripeptide, and free Lys. 2,4-Dinitrophenyl-histidine was recovered from
the pentapeptide, and 2,4-dinitrophenylphenylalanine was recovered
from the tripeptide.
5. Digestion with the enzyme pepsin produced a dipeptide, a tripeptide, and
a tetrapeptide. The tetrapeptide was composed of (Lys)2, Phe, and Gly.
The native sequence was determined to be: