Amino Acids and Proteins
3-D Structure of Myoglobin
Importance of Proteins
• Main catalysts in biochemistry: enzymes (involved in
virtually every biochemical reaction)
• Structural components of cells (both inside and
outside of cells in tissues)
• Regulatory functions (if/when a cell divides, which
genes are expressed, etc.)
• Carrier and transport functions (ions, small
molecules)
Levels of Protein Structure
• Primary Structure - amino acid sequence in a
polypeptide
• Secondary Structure - local spatial arrangement of a
polypeptide’s backbone atoms (without regard to side
chain conformation)
• Tertiary Structure - three-dimensional structure of
entire polypeptide
• Quaternary Structure - spatial arrangement of
subunits of proteins composed of multiple
polypeptides (protein complexes)
Structure of -amino acids
The 20 Amino Acids Found in Proteins
Properties of Different Amino Acid Side
Chains
Stereochemistry of -amino acids
Stereoisomers of -amino acids
All amino acids in
proteins are L-amino
acids, except for
glycine, which is
achiral.
RS Nomenclature System (Cahn, Ingold,
Prelog System)
Alternative Representation of Amino Acids
All L-amino acids in proteins are S, except for
cysteine, which is R.
Leucine
Cysteine
CH3
H C CH3
CH2
H3N C COO
H
SH
CH2
H3N C COO
H
=
H3N
(S)
COO
H3N
(R)
COO
=
H3N
(S)
COO
SH
=
=
H3N
(R)
COO
SH
Properties of Cysteine Side Chain
pKa = 8.2
SH
S
+
H 3N
H3N
COO
H3N
COO
COO
H3N
COO
oxidation
+ 2H+
HS
S
S
SH
reduction
H3N
H3N
H+
Side chains with -SH or
-OH can ionize, making
them more nucleophilic.
+ 2e-
Oxidation between pair of
cysteine side chains results
in disulfide bond formation.
COO
COO
Disulfide bonds are mainly found in extracellular proteins; the ~5 mM
glutathione (g-Glu-Cys-Gly) makes the inside of the cell a highly
reducing environment.
Hydroxyl-Containing Amino Acid Side Chains
pKa = 13
OH
Serine
H 3N
COO
HO
CH3
O
H3N
+
H+
+
H+
COO
pKa = 13
Threonine
H 3N
O
COO
H3N
OH
CH3
COO
pKa = 10.1
O
Tyrosine
+
H 3N
COO
H 3N
COO
H+
Tyrosine, Serine and Threonine Can Be
Phosphorylated in Proteins
Example: Tyrosine
:Base-Enzyme (Kinase)
O H
N
H
O
Tyrosyl Residue in a Protein
O
NH2
N
O
+
N
H
O
O
O
O
O P O P O P O
O
O
O
Mg2+
N
O
OH OH
O
P
O
O
N
N
N
H
O
Phosphotyrosyl Residue
+ 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.
pI of Glu = (2.19 + 4.25)/2 = 3.22
pI of His = (6.0 + 9.17)/2 = 7.59
Formation of a Peptide
Planarity of Peptide (Amide) Bond
.
cis and trans Isomers
The trans isomer is generally more
stable because of steric crowding
of side chains in the cis isomer.
Examples of Oligopeptides
N- and C-Termini May Be Modified in
Proteins
Primary Structure of Bovine Insulin
First protein to be fully sequenced (by
Fred Sanger in 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
Translation elongation factor Tu/1
Myoglobin
The Genetic Code
DNA
RNA
Protein
Initiating Amino Acid in Translation
CH3
S
O
H
O
H
N
N
H
O
N-Formylmethionine in
prokaryotes
R
CH3
S
O
H
N
H 3N
O
R
Just methionine in
eukaryotes
Charging of tRNAs with Specific Amino
Acids
Translation of mRNA into Protein
Ribosomal Peptidyl Transferase Activity
Note: the catalytic component of the ribosome’s peptidyl transferase activity is
RNA; it’s an example of a catalytic RNA or ribozyme.
Disulfide Bond Formation in Insulin
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 isolectric point 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 fixedangle rotor (pellet at bottom of tube vs.
supernatant solution above)
•Zonal ultracentrifugation (e.g., sucrosegradient) – swinging-bucket rotor
•Equilibrium-density gradient
ultracentrifugation (e.g., CsCl) –
swinging-bucket or fixed-angle rotor
Zonal Centrifugation: Sucrose-Gradient
Preparative Ultracentrifugation
Separates by sedimentation coefficient (determined
by size and shape of solutes)
Sucrose-Gradient Preparative
Ultracentrifugation
Equilibrium Density Gradient
Ultracentrifugation
•
•
•
•
Used in Meselsen-Stahl experiment.
Separates based on densities of solutes.
Does not require premade gradient.
Pour dense solution of rapidly diffusing substance in
tube (usually CsCl).
• Density gradient forms during centrifugation (“selfgenerating 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/molecular sieve - 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., 6 M 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
Identification of N-Terminal Residue
NO2
Note: Identification of C-terminal residue done by hydrazinolysis (reaction with anhydrous hydrazine in presence of mildly
acidic ion exchange resin) or with a C-terminus-specific exopeptidase (carboxypeptidase).
Separation of Amino Acids by HPLC
Protein Identification by Mass
Spectrometry
Protein Identification by Mass
Spectrometry
Two main approaches:
1. Peptide mass fingerprinting: Proteolytic digestion of protein,
then determination m/z of peptides by MS (e.g., MALDI-TOF
or ESI-TOF), search “fingerprint” against database. Success
of ID depends on quality/ completeness of database for
specific proteome.
2. Tandem MS (MS/MS – e.g., nanoLC-ESI-MS/MS):
Proteolytic digestion of protein, separation and determination
of m/z of each (MS-1), then determination of collision-induced
dissociation fragment spectrum for each peptide (MS-2).
Gives context/sequence-dependent information, so more of a
do novo sequencing method.
Locating Disulfide Bonds
O
I
Oiodoacetate
Determing Primary Structure of an Entire
Protein
Reactions in Solid-Phase Peptide
Synthesis