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Chapter 3
Amino Acids, Peptides, and Proteins
DNA RNA Protein • Posttranslationally modified proteins• Protein-ligand interactions
Biological phenomena
Transcription Translation
ReverseTranscription
Central Dogma in Biological System
Specific covalent sequences of monomers
Noncovalent Interactions1. Hydrogen bonds2. Ionic Interactions3. Hydrophobic Interactions4. van der Waals interactions
Correct three-dimensional structures
Biological functions
Amino Acids
Amino Acids
20 Amino acids Numbering of carbons
… from C bonded to NH3
+ and COO-
Chiral molecule (exp. Gly) -Carbon is a chiral center Stereoisomer; enantiomer
1
2
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
Cell wall (plant) Collagen (connective tissue)
Collagen
Myosin
Prothrombin (blood clotting)
Ca2+ binding proteins Elastin
A few proteins Incorporation
during translation
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 -amino group
One free -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
22
Conjugated Proteins
[1] Simple Protein amino acids
[2] Conjugated Proteinamino 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
Genomics(Structural)
FunctionalGenomics
Proteomics (St./Fn.)
DNA 염기서열
약 30 억개(2-3% 유전자 )
유전자 및 기능파악
총 2 만 5 천 유전자 추정현재까지 9,000 여종 파악
단백질의 특성 및기능 파악
조직별 5,000 - 20,000 추정
Postgenomic era
Proteomics
[1] Biological Perturbation
[2] High-throughput Screening
two-dimensional gel electrophoresisin-gel protein digestionMALDI-TOF mass spectrometrypeptide 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