Chapter 3
Amino Acids, Peptides,
and Proteins
Central Dogma in Biological System
Transcription
DNA
Translation
RNA
Protein
Reverse
Transcription
• Posttranslationally
modified proteins
• Protein-ligand
interactions
Biological phenomena
Specific covalent sequences of monomers
Noncovalent Interactions
1. Hydrogen bonds
2. Ionic Interactions
3. Hydrophobic Interactions
4. van der Waals interactions
Correct three-dimensional structures
Biological functions
Amino Acids
Amino Acids
 20 Amino acids
 Numbering of carbons

a , b, g… from C bonded to NH3+
and COO-
 Chiral molecule (exp. Gly)


a-Carbon is a chiral center
Stereoisomer; enantiomer
1
2
a
Classification of Amino Acids
UV absorption at 280 nm
Absorption of light by molecules
Classification of Amino Acids
• Nonpolar
• Structural role
Classification of Amino Acids
Uncommon Amino Acids
 Collagen
 Myosin
 Cell wall (plant)
 Collagen (connective tissue)
 A few proteins
 Incorporation
during translation
 Prothrombin (blood clotting)
 Ca2+ binding proteins
 Elastin
Reversible amino acid modifications
Amino Acids as Acids and Bases
 Zwitterion
 Either acids or bases
 Ampholytes (substances with zwitterionic nature)
Titration of Amino Acids
 Two pKa and two
buffering regions
 pI (isoelectric point or
isoelectric pH)



Characteristic pH with zero
net electric charge
Above pI : negative charge
Below pI : positive charge
 pI = (pK1 + pK2)/2 = 5.92
Effect of Chemical Environment on pKa
Amino Acids with Ionizable R Group
 pI = (pK1 + pKR)/2 = 3.22
 pI = (pKR+ pK2)/2 = 7.59
Peptides and Proteins
Peptides and Proteins
 Peptide; Chains of amino acids

Dehydration reaction b/t amino acids peptide bond
 Polypeptide vs. protein
 Polypeptide: Mr<10,000
 Amino-terminal (N-terminal)
 Carboxyl-terminal (C-terminal)
Ionization of Peptide
 Ionization of peptide
 One free a-amino group
 One free a-carboxyl group
 Inonizable R groups
 pKa of R groups in peptide
 Different from pKa of free amino
acid  affected by environmental
factors
Biologically Active Peptides and
Polypeptides
 Size
 Small peptide
 Vertebrate hormones (peptide hormones)


 Oxytocin (9), thyrotropin-releasing factor (3), insulin (30 + 21)
Antibiotics, amanitin
Most of the proteins
 < 2,000 a.a. (exception, titin)
 Oligomeric status
 Single polypeptide chain
 Multisubunit proteins (non-covalent interaction)
 Oligomeric : at least two subunits are identical
 Protomers : identical units
 Calculation of the number of amino acid residues
 Mr / 110
 Average Mr of 20 a.a. : 138
 Average Mr of protein a.a : 128
 Removal of water during peptide bond formation : 128 -18 =110
Hemoglobin
a 2b 2
Conjugated Proteins
[1] Simple Protein
amino acids
[2] Conjugated Protein
amino acids + prosthetic groups
Working with Proteins
Protein Purification
 Cell lysis (optionally differential centrifugation)

Crude extract
 Fractionation
 Use differences in protein solubility
 Depending on pH, temperature, salt
concentration etc.
 Salting out
 Addition of ammonium sulfate ((NH4)2SO4) for
differential precipitation of proteins
 Dialysis

Exchange of salts and buffer using semipermeable
membrane (e.g. removing (NH4)2SO4)
 Column chromatography


Separation of proteins based on charge, size,
binding affinity etc.
Stationary phase vs.
Mobile phase containing proteins
Ion-exchange chromatography
 Cation-exchange chromatography
 Solid phase : negatively charged group
 Positive charged proteins migrate
slowly
 Anion-exchange chromatography
 Solid phase : positively charged group
 pH & salt concentration
 affect protein affinity on solid matrix
 Separation by pH or salt gradient
 Other cautions
 Diffusional spreading
 expansion of protein band
Size-Exclusion Chromatography
 Solid phase : cross-linked polymer
beads with engineered pores or
cavities of a particular size
 Small proteins enter the pores
 Slow migration
Affinity Chromatography
 Beads with covalently attached chemical group
 Binding of proteins with affinity for the chemical
group
Protein Purification
 HPLC (high-performance liquid chromatography)
 Use high pressure pump that speed the movement
of the protein molecules
 Limited diffusion  High resolution
 Determining the methods for protein purification
 Mostly empirical
Separation & analysis of Protein by
Electrophoresis
 Electrophoresis



Separation of charged proteins in an electric field
Electrophoretic mobility of proteins
 Depending on size and shape of proteins
Advantages
# of different proteins, purity of protein
preparation, determination of pI & mw
 SDS-polyacrylamide gel electrophoresis
Determining Molecular Weight of a
Protein
SDS PAGE
(polyacrylamide gel electrophoresis)
Isoelectric Focusing
 Procedure to determine the pI of a protein


Establishment of pH gradient
 Gel containing a mixture of low molecular weight organic acids
and bases (ampholytes) with different pI value
 Application of electric field
Each protein migrates until it reaches the pH corresponding to its pI
Two-Dimensional Electrophoresis


1st : Isoelectric focusing
2nd : SDS-PAGE
Postgenomic era
Genomics
(Structural)
Functional
Genomics
DNA 염기서열
유전자 및 기능파악
약 30억개
(2-3% 유전자)
총 2 만 5천 유전자 추정
현재까지 9,000여종 파악
Proteomics
(St./Fn.)
단백질의 특성 및
기능 파악
조직별
5,000 - 20,000 추정
Proteomics
[1] Biological Perturbation
[2] High-throughput Screening
two-dimensional gel electrophoresis
in-gel protein digestion
MALDI-TOF mass spectrometry
peptide mass mapping
[3] Bioinformatics
[4] Conventional Protein Chemistry
Investigation of proteins with
Mass spectrometry
 Components of mass spectrometer
 Ionizer: converting molecules to gas phase ions
 Soft ionizer for large molecules
 MALDI (matrix-assisted laser desorption/ionization) MS
 ESI (electrospray ionization) MS
 Mass analyzer: separate the ions according to the
m/z
 Time of flight (TOF)
 Measuring the time take by ions to travel to the detector
 Ion detector
 Mass spectrometer for protein analysis



Small amount of protein (extraction from 2D-gel)
Determination of molecular weight
Determination of short polypeptide sequence
 Tandem MS or MS/MS
MALDI-TOF
 Matrix-assisted laser desorption/ionization
mass spectrormetry
 Protein placed in a light-absorbing matrix
 Ionization and desorption of proteins by a
short pulse of laser
ESI-TOF
 Electrospary ionization mass spectrometry
 Passing of analyte solution through a charged
needle with a high electrical potential
 Dispersion of charged microdroplets (fine mist)
Tandem MS
The Structure of Proteins
Determination of Amino Acid
Sequence
 Determination of amino acid sequence from a
protein
 Sanger’s method
 N-terminal labeling and identification
 Using FDNB (1-fluoro-2,4-dinitrobenzene)
 Edman Degradation
 Sequencing of the entire polypeptide
 Sequential labeling and removal of the N-terminal amino
acid
 Sequenator
 Automated sequencing of proteins
 Accuracy is depending on the efficiency of the individual
chemical step
 > 99% in modern sequenator
 Translation from DNA sequence
 DNA sequence  protein sequence
 Protein sequence  cloning of the gene
Determination of Amino Acid
Sequence
Sanger
Edman
Sequencing Large Proteins
 Breaking disulfide bonds
 Oxidation by performic acid
 Reduction and carboxymethylation
 Cleaving the polypeptide chain
 Using proteases
 Cleavage of peptide bond next to particular amino
acid residues
 Trypsin: Lys, Arg
 Sequencing of peptides
 Ordering peptide fragments
 Compare sequences generated from different
cleavage methods
 Locating disulfide bonds
 Comparison of cleavage fragment with or without
breaking disulfide bonds
Breaking Disulfide Bonds
Sequencing Large Proteins
Chemical Synthesis of Small Peptide
 Developed by R. Bruce Merrifield (1962)
 Synthesis from C- to N- terminal on an polymer
support
 Fmoc (9-fluorenylmethoxycarbonyl)
 Protection of unwanted reaction
Chemical Synthesis of Small Peptide
Chemical Synthesis of Small Peptide