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