Chapter 3 Amino Acids, Peptides, and Proteins

DNARNAProtein Posttranslationally modified proteins Protein-ligand interactions Biological phenomena TranscriptionTranslation Reverse Transcription Central Dogma in Biological System 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

20 Amino acids Numbering of carbons   … from C bonded to NH 3 + 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) Ca 2+ 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 = (pK 1 + pK 2 )/2 = 5.92

Effect of Chemical Environment on pKa

Amino Acids with Ionizable R Group pI = (pK 1 + pK R )/2 = 3.22 pI = (pK R + pK 2 )/2 = 7.59

Peptides and Proteins

Peptide; Chains of amino acids  Dehydration reaction b/t amino acids  peptide bond Polypeptide vs. protein  Polypeptide: M r <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 pK a of R groups in peptide  Different from pK a 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 ( )  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  M r / 110  Average M r of 20 a.a. : 138  Average M r of protein a.a : 128  Removal of water during peptide bond formation : =110

Hemoglobin 222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 ((NH 4 ) 2 SO 4 ) for differential precipitation of proteins Dialysis  Exchange of salts and buffer using semipermeable membrane (e.g. removing (NH 4 ) 2 SO 4 ) 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  1 st : Isoelectric focusing  2 nd : SDS-PAGE

Genomics (Structural) Functional Genomics Proteomics (St./Fn.) DNA 염기서열 약 30 억개 (2-3% 유전자 ) 유전자 및 기능파악 총 2 만 5 천 유전자 추정 현재까지 9,000 여종 파악 단백질의 특성 및 기능 파악 조직별 5, ,000 추정 Postgenomic era

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