Chapter 3

Exploring Proteins and Proteomes

• A collective name for the genes existed in an organism

• C. elegance (roundworm) : 97 million bases, 19,000 genes

Drosophila melanogaster (fruit fly) : 180 million bases, 14,000 genes

Human : 3 billion bases, ~20,000 genes

• Static and absolute information

Genome

• A collective name for the proteins expressed by the genome

• Dynamic and functional information

• It varies with cell type, developmental stage, and environmental condition

such as the presence of hormones.

• Regulation of mRNA synthesis, alternative splicing, mRNA stability, rate of

protein synthesis, post-translational modification, protein stability control,

protein degradation

Proteome

• The purification of proteins is an essential first step in understanding their

function.

• Purification should yield a sample of protein containing only one type of

molecule of interest.

• Proteins can be separated from one another on the basis of solubility, size,

charge, and binding ability.

• Assay : a test for some unique identifying property of the protein

• Specific Activity : the ratio of enzyme activity to the amount of protein in the

enzyme assay

Protein Purification

NADHcan absorb light

at 340 nm.

Nicotinamide adenine dinucleotide

Homogenation and Fractionation by Centrifugation

• Salting Out : protein solubility decrease by very high concentration of salt

• Salting In : protein solubility increase by low concentration of salt

• Dialysis : separation of small molecules from proteins through membrane with

pores such as cellulose membrane (cf. semi-permeable)

Salting Out & Dialysis

Ammonium sulfate for protein precipitation

Gel-Filtration Chromatography(Molecular Exclusion,

Size Exclusion,Molecular Sieve)

1. Thyroglobulin (669 kd)

2. Catalase (232 kd)

3. BSA (67 kd)

4. Ovalbumin (43 kd)

5. Ribonuclease (13.4 kd)

Anion ExchangerPositively Charged Column

Negatively Charged Proteins

Cation ExchangerNegatively Charged ColumnPositively Charged Proteins

Ion-ExchangeChromatography

Depend on local charge on proteins

LCLiquid Chromatography

FPLCFast Pressure Liquid Chromatography

HPLCHigh Pressure Liquid Chromatography

AffinityChromatography

(concanavalin A)

Highly specific- Histidine tag

Gel Electrophoresis

v = Ez / fv : velocity of migrationE : electric field strength

z : net charge on the proteinf : frictional coefficient

f = 6r : viscosity of the medium

r : radius of the protein

Polyacrylamide gel electrophoresis

Polymer Formation of Acrylamide using Bis-Acrylamidefor PAGE (Poly-Acrylamide Gel Electrophoresis)

AmmoniumPersulfate

Sieving action!

SDS-PAGE : Denaturing Gel(Determination of the Molecular Weight of Protein)

Coomassie Blue Staining; > 0.1 g)(cf. Silver Staining: > 0.02 g)

Under BME, DTT

One SDS anion for every two a.a.

Except carbohydrate-rich proteins, membrane proteins

Mobility ; Log of MWResolution: 2% MW difference

Isoelectric Focusing & Two Dimensional Electrophoresis

pI : Isoelectric Point (pH with net charge zero)

Evaluation of Protein Purification

As purification continues, relative presence of

contaminants should be decreased and

the proportional amount of the protein of

interest should be increased.

Centrifugation & Sedimentation Coefficient

• A more massive particle sediments more rapidly.

• A more compact shaped particle sediments faster.

(i.e. elongated particles sediments more slowly than do

spherical ones of the same mass. Frictional coefficient

f)

• A denser particle sediments more rapidly. Buoyant

force is smaller for the denser particle

• v < 1 : sink, v > 1 : float, v = 1 : no movement

s = m(1 - v) / fs : sedimentation coefficient

m : mass of the particle

v : partial specific volume; the reciprocal of the particle density : density of the medium

(1 - v) : buoyant force exerted by liquid medium

f : frictional coefficient; a measure of the particle shape

a. Seidmentation process in the cell

From Archimedes’ principleBuoyancy = weight of displaced fluid

Or fluid density x submerged vol x g

Principle of Analytical Ultracentrifugation

b.solute distribution in the cell

S Value for Various Proteins

Density and Sedimentation Coefficient

for Various Cellular Components

Gradient (Zonal or Band) Centrifugation:

Separation of Non-Denatured Proteins with different sedimentaion coefficients

(Size, Density and Shape)

Sedimentation equilibrium: centrifuged at low speed so that sedimentation is counter balanced by diffusion-Very accurate in mass determination without denaturing.- useful for large multimeric proteins.

Sedimentation velocity

Determination of Amino Acid Composition of the Peptide

1. Peptide hydrolyzation by heating it in 6N HCl at 100oC for 24 hrsAla-Gly-Asp-Phe-Arg-Gly

(Asp, Gly2, Ala, Phe, Arg)

2. Separation of amino acid hydrolysates by ion-exchange chromatography (e.g. sulfonated polystyrene resin; Dowex-50)

3A. Quantitation of Each Fraction by Ninhydrin;Yield Visible Color (usually blue except Pro for yellow);Detection Sensitivity = Microgram (10 nmol) of an Amino Acid

3B. Quantitation of Each Fraction by Fluorescamine;Yield Fluorescence;Detection Sensitivity = Nanogram (10 pmol) of an Amino Acid

Identification of N-Terminal Amino Acid

(FDNB) YieldFluorescentSulfonamide

FDNB, Dabsyl Chloride, or Dansyl Chloride

Can Specifically React with the N-terminal Amino Group, and

Yield DNB-Amino Acid, Dabsyl Amino Acid, or Dansyl Amino Acid, and

These Can Be Identified by Their Chromatographical Properties.

Determination of Amino Terminal Residue of a Peptideusing Dabsyl Chloride

Edman Degradation Sequentially Removes One Residue at a Timefrom the Amino End of a Peptide up to 50 times

Each round

can be complete

within 1 hr and

the Edman degradation

can be repeated

up to 50 cycles

in Practice.

Phenyl Isothiocyanate (PITC) Can Specifically React with

the N-terminal Amino Group, and Yield Phenyl Thiocarbamoyl (PTH) Amino Acid,

and This Can Be Identified by Its Chromatographical Property.

Separation of PTH-Amino Acids

Current Sensitivity ofPTH-AA Detection UsingGas-Phase Sequenator:

Picomole

Mild acidic condition

For sequencing of an entire Protein…??Divide and Conquer !!!

Sequence Specific Cleavage by Cyanogen Bromide

Sequence Specific Cleavage by Trypsin

Deduction of Full Amino Acid Sequence of a Proteinby Overlapping the Sequences Obtained from individual Peptides

Reduction of Disulfide Bonds :

-Mercaptoethanol, Dithiothreitol (DTT)

Sequencing of Multimeric Proteins

Denaturation :

Urea, Guanidium Chloride, SDS

Preservation of Reduced Sulfhydryl Groups :

Alkylation using Iodoacetate

Separation of Each Polypeptide Chain :

SDS-PAGE

Diagonal Electrophoresisto Determine the Positions of the Disulfide Bonds

Reduction and Oxidation of Disulfide Bonds by Performic Acid

The Amino Acid Sequence Provides Insightsinto the Protein’s Function, Structure, and History

1. The sequence of a protein of interest can be compared with all other known

sequences to ascertain similarities. (Family, function prediction possible)

2. Comparison of sequences of the same protein in different species yields a wealth

of information about evolutionary pathway.

3. Amino acid sequences can be searched for the presence of internal repeats.

4. Many proteins contain amino acid sequences that serve as signals designating

their destinations or controlling their processing. (N-terminal 20 hydrophobic

residues, signal sequence, nuclear localization signal)

4 Repeating Motifs

in Calmodulin :

Each Unit Binds

a Calcium Ion

• Amino acid sequence data provide a basis for preparing antibodies specific for a

protein of interest.

• Amino acid sequence are valuable for making DNA probes that are specific for the

genes encoding the corresponding proteins.

• The nucleotide sequence of DNA (gene) directly reveals the entire amino acid

sequence of the protein encoded by the gene.

• However, DNA sequence can not disclose the information regarding post-translational

modification.

Practical Usage of Amino Acid and DNA Sequences

Antibody

• Antibody (immunoglobulin) is a protein synthesized by an animal in response to the

presence of a foreign substance (antigen).

• Antibodies have specific and high affinity against antigens.

• Proteins, polysaccharides and nucleic acids can be effective antigens.

• Epitope : a specific group or cluster (portion) of antigen to stimulate the synthesis of an

antibody and recognized by a specific antibody (antigenic determinant)

• Hapten : a small molecule containing epitope attached to a carrier

Antibody (continued)

• Each antibody producing cell synthesizes only one type of

antibody recognizing a single kind of epitope.

• The proliferation of a given antibody producing cell is

stimulated by the binding of its designated antigen to the cell

surface receptor of the antibody producing cell .

• Periodic injections of an antigen into the host animal can raise

the antibodies specifically recognizing the injected foreign

substance.

• Blood withdrawn from the immunized host animal

centrifugation separation of blood cells (pellet) and serum

(supernatant) anti-serum

• Anti-serum contains multiple kinds of antibodies each

recognizing a different surface feature of the same antigen.

• This heterogenic antibodies are called as polyclonal antibodies.

• This heterogeneity can complicate the use of these antibodies.

Monoclonal Antibody

• Monoclonal hybridoma cell lines can generate large amount of homogeneous antibodies.

• Monoclonal antibodies can serve as precise analytical, preparative and therapeutic reagents.(HCV, HIV, herceptin)

Immuno-Staining ofDrosophila

Embryousing

MonoclonalAntibodyagainst

Engrailed

Plasma cell

by antigen-antibody interaction

Monoclonal antibody drugs?

Herceptin Binds to the C-terminus of Domain IV

Herceptin Fab

I

III

II

IV

N

C

HER2

Ribbon Diagram of Her-3 ECD

N

C

“Tethered”I

II

III

IV

Right-handed helixLaminin-like folds

Fig. 14.28 pp397

Surface representations of EGFR and HER2 in Antibody-Bound Conformations

Herceptin

ELISA (Enzyme-Linked Immuno-Sorbent Assay)

Antibody detection, anti-HIV antibody

Antigen detection

Western Blotting

Radioactive secondary antibody

For protein expression and purification

Immuno-FluorescenceMicroscopy

Actin Filament Stainingusing -actin antibody

Immuno-ElectronMicroscopy

Detection of a channel proteinfrom the synaptic vesicles

using antibodies tagged withelectron-dense markers such as gold or

ferritin(Resolution better than 10 nm)

Fluorescence-labeled antibodies(resolution 200nm)

ex) Glucocorticoid receptor

Synthetic Peptides

• Synthetic Antigens for antibody formation• Receptor or Interacting Protein Isolation• Clinical Drugs (ex, vasopressin)• 3D Structure Study

• Increase water absorption in the kidney; Aquaporin • Caffeine decreasesrelease of AVP

Protection of Amino Groupby t-BOC

Activation of Carboxyl Groupby DCC

Solid Phase

Peptide Synthesis

(more than 100 amino acids)

Resin : Insoluble Matrix (Polystyrene Beads), HF : Hydrofluoric Acid

MALDI-TOFMass

Spectrometry

MALDI : Matrix-AssistedLaser Desorption-Ionization

TOF : Time of FlightF=ma

Mass Spectrometry

Are Often Combined with

2D Electrophoresis

for Proteome Analysis

h

Laser

+20 kV

Variable Ground Grid Grid

AH+

Sample plate 1. Sample (A) is mixed with excess matrix (M) and dried on a MALDI plate.

2. Laser flash ionizes matrix molecules.

3. Sample molecules are ionized by proton transfer from matrix:

MH+ + A M + AH+.

Why MALDI?

-Less sensitive to salts-Lower PRACTICAL detection limits-Easier to interpret spectra(less multiple

charges)-Quick and easy-Higher mass detection-Higher Throughput(1000>samples per

hour)

The Mass Analyzer: TOFTime Of Flight(TOF)

Flight TubeIon Source

Principle: If ions are accelerated with the same potential at a fixed point and a fixed initial time and are allowed to drift, the ions will

separate according to their mass to charge ratios.

20-25 kV

++

• Peptide can be sequenced by MS

Individual proteins can be identified by MS

• Protein cleavage, followed by chromatographic separation and MS

NMR (Nuclear Magnetic Resonance)

• Basis for Resolution : Electron (Proton) Chemical Shift in Magnetic Field

• Advantage

- Solving Protein Structures in Solution State

- Requires Smaller Quantity for Analysis than X-ray Crystallography

• Limitations

- Technical Difficulties for Bigger Size Proteins (> 40 kD)

- Technical Difficulties for Multi-Subunit Proteins

X-Ray Crystallography• Basis for Resolution : X-Ray Diffraction and Electron Scattering

• Advantage

- No Size Limitation for Analysis

- Solve the Structure of Multi-Subunit Complexes

• Limitations

- Solving Protein Structures in Solid State

- Difficulties in Making Protein Crystals with Good Diffraction Properties