ASAM AMINO DAN PROTEIN

Struktur 3-D dari Mioglobin

 Kegunaan Protein

• Katalis utama dalam biokimia: enzim (terlibat dalam

setiap reaksi biokimia)
• Komponen struktur dari sel (baik di dalam maupun di
luar sel dalam jaringan )
• Fungsi Regulator (if/when a cell divides, which
genes are expressed, etc.)
• Fungsi Carrier and transport (ion, molekul-molekul
kecil)
• Protein cadangan
• Protektif
• Bagian dari makanan sebagai pengganti jaringan sel
 Tingkatan Struktur Protein

• Struktur Primer – Urutan asam amino dalam

polipetida
• Struktur sekunder – Hubungan spasial semua atom
terhadap semua atom lainnya dalam polipeptida
• Struktur Tersier – Struktur 3 dimensi dari seluruh
polipetida
• Struktur Quaternar - susunan spasial subunit dari
protein yang tersusun dari multiple polypeptide
(protein complexes)
Structure asam -amino
The 20 Asam Amino dalam Protein
PI = (pK1 + pK2) / 2
Properties of Different Amino Acid Side
Chains
Stereochemistry of -amino acids
Stereoisomers of -amino acids

All amino acids are

chiral except glycine.

All amino acids in

proteins are L-amino
acids.
RS Nomenclature System (Cahn, Prelog,
Ingold System)

Penurunan prioritas

SH > OR > OH > NHCOR >

NH2 > COOH > CHO > CH2OH
> C6H5 > CH3 > H
Alternative Representation of Amino Acids

H
Glycine H3N+ C COO- = H3N+ COO-
H

All L-amino acids in proteins are S-amino

acids, except for cysteine, which is R.

CH3 H3C H3N+(S) COO-

=
H3C CH CH3 =
Leucine CH2 CH3
H3N+ C COO- H3N+(S) COO- H3C
H

SH SH H3N+(R) COO-
Cysteine CH2 = =
H3N C COO-
+
H3N+(R) COO-
H SH
 Properties of Cysteine Side Chain

pKa = 8.3 S-
Side chains with -SH or
SH
CH2 -OH can ionize, making
CH2 + H+
H3N C COO-
+
them more nucleophilic.
H3N C COO-
+
H
H

H
H3N+ C COO-
H
CH2
oxidation H3N+ C COO- Oxidation between pair of
SH
CH2 + 2H+ cysteine side chains results
S
S + 2e- in disulfide bond formation.
SH
reduction CH2
CH2
H3N C COO-
+
H3N C COO-
+
H
H
Hydroxyl-Containing Amino Acid Side Chains
pKa = 13
OH O-
CH2 CH2 + H+
Serine H3N C COO-
+
H3N+ C COO-
H H

CH3 pKa = 13 CH3

HC OH HC O-
+ H+
Threonine H3N+ C COO- H3N+ C COO-
H H

OH pKa = 10.1 O-

Tyrosine + H+
CH2 CH2
H3N+ C COO- H3N+ C COO-
H H
 Tyrosine, Serine and Threonine Can Be
Phosphorylated in Proteins
Example: Tyrosine

OH :Base-Enzyme (Kinase)

N O O-
H
O P
O O-
Tyrosyl Residue in a Protein

NH2
O- N
N
N
N H
O O O N O
+ -O P O P O P O O Phosphotyrosyl Residue
O- O- O-
N OH OH
H
O
ATP + ADP
Modified or Unusual Amino Acids
Absorption of UV Light by Aromatic Amino
Acids
Titration of Amino Acids with Ionizing Side
Chains

Isoelectric point (pI) for amino acids with ionizable

side chains:
Take average pKa for the two ionizations involving the
neutral (net charge of zero) species.
Formation of a Peptide
Planarity of Peptide (Amide) Bond

Quick Time™a nd a TIFF ( Uncomp res sed) deco mpre ssor are n eede d to s ee this picture .
cis and trans Isomers
Examples of Oligopeptides
N- and C-Termini May Be Modified in
Proteins
Primary Structure of Bovine Insulin

First protein to be fully sequence;

Fred Sanger, 1953. For this, he won his first
Nobel Prize (his second was for the Sanger
dideoxy method of DNA sequencing).
Evolution and Conservation of Protein
Sequences
The Genetic Code
DNA RNA Protein
Initiating Amino Acid in Translation

CH3
S N-Formylmethionine in
O O prokaryotes
H
N
H N
H
O R

CH3
S

O
Just methionine in
H eukaryotes
N
H3N+
O R
Charging of tRNAs with Specific Amino
Acids
Translation of mRNA into Protein
Post-Translational Modification of Proteins
Methods in Protein Biochemistry
Gel Electrophoresis
 Polyampholyte Character of a
Tetrapeptide and Isoelectric Points

Group pKa
-NH3+ 9.7
Glu g-COOH 4.2
Lys e-NH3+ 10.0
-COOH 2.2

Isoelectric Point (pI), pH at

which molecule has net zero
charge, determined using
computer program for known
sequence or empirically (by
isoelectric focusing)
Isoelectric Focusing
Electrophoresis through polyacrylamide gel in
which there is a pH gradient.
 Two-Dimensional Gel Electrophoresis

• Separate proteins based on pI in 1st dimension

• Separate proteins based on molecular weight in 2nd
dimension
 “Salting Out”: Ammonium Sulfate
Precipitation in Protein Fractionation
Centrifugation
Low-speed, high-speed, or
ultracentrifugation: different
spin speeds and g forces

Centrifugation Methods

•Differential (Pelletting) – simple method

for pelleting large particles using fixed-
angle rotor (pellet at bottom of tube vs.
supernatant solution above)

•Zonal ultracentrifugation (e.g. sucrose-

gradient) – swinging-bucket rotor

•Equilibrium-density gradient
ultracentrifugation (e.g. CsCl) –
swinging-bucket or fixed-angle rotor
Zonal Centrifugation: Sucrose-Gradient
Preparative Centrifugation

Separates by sedimentation coefficient (determined

by size and shape of solutes)
Sucrose-Gradient Preparative
Centrifugation
 Equilibrium Density Gradient
Ultracentrifugation

• Used in Meselsen-Stahl experiment

• Separates based on densities of solutes
• Does no require premade gradient
• Pour dense solution of rapidly diffusing substance in
tube (usually CsCl)
• Density gradient forms during centrifugation (“self-
generating gradient”)
• Solutes migrate according to their buoyant density
(where density of solute = density of CsCl solution)
Column Chromatography

Flow-through Eluate
 Different Types of Chromatography

• Gel filtration/size exclusion - separates by size (molecular

weight) of proteins
• Ion exchange (cation exchange and anion exchange) -
separates by surface charge on proteins
– Cation exchange: separates based on positive charges of
solutes/proteins, matrix is negatively charged
– Anion exchange: separates based on negative charges of
solutes/proteins, matrix is positively charged
• Hydrophobic interaction - separates by hydrophobicity of
proteins
• Affinity - separates by some unique binding characteristic of
protein of interest for affinity matrix in column
Ion-Exchange Chromatography
Gel Filtration Chromatography
Affinity Chromatography
 Cleavage of Polypeptides for Analysis

• Strong acid (e.g. 6M HCl) - not sequence specific

• Sequence-specific proteolytic enzymes (proteases)
• Sequence-specific chemical cleavage (e.g. cyanogen
bromide cleavage at methionine residues)
Protease Specificities
Cyanogen Bromide Cleavage at
Methionine Residues
Protein Sequencing: Edman Degradation

PTC = phenylthiocarbamyl

F3CCOOH = trifluoroacetic acid

PTH = phenylthiohydantion
Separation of Amino Acids by HPLC
Protein Identification by Mass
Spectrometry
Locating the Disulfide Bonds in Insulin

O
I
O-
iodoacetate
Determing Primary Structure of an Entire
Protein
Reactions in Solid-Phase Peptide
Synthesis