David L. Nelson and Michael M. Cox

Lehninger Principles of

David L. Nelson and Michael M. Cox

Lehninger Principles of BiochemistryFourth EditionFourth Edition

Chapter 3:Chapter 3:

Amino Acids, Peptides, and Proteins

Copyright © 2004 by W. H. Freeman & Company

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hemoglobinsFirefly light by luciferase, Keratin : rhinoceros horn, hemoglobinsFirefly light by luciferase,

luciferins, ATP

Keratin : rhinoceros horn, nail etc

Structure of AAStructure of AA

• All α aa– Cα linked 3 groups– Cα linked 3 groups

• An amino• A carboxyl• A R group:side• A R group:side• Normally 20 but some

more but not as structural, for special funciton in specific proteinsspecific proteins

– Assigned • 3-letter abbreviations • 1 letter symbol• 1 letter symbol

– Addtion carbon to C α γ�β∂βε or 1.2(C α).3.4.5

4 different groups

Cα� chiral centerCα� chiral center

2 unique spatial arrangments

2 stereoisomers2 stereoisomers

Nonsuperimposable mirror

Enantiomers

Optical active

L & D forms

All (almost) � L

hydr

ophi

l

h

ydro

phob

hydr

ophi

l

h

ydro

phob

hydr

ophi

l

h

ydro

phob

hydr

ophi

l

h

ydro

phob

hydr

ophi

l

h

ydro

phob

Ala. Val. Leuc and Ala. Val. Leuc and isoleucine clustering

At pH 7

All hydrophobic interaction

Tyrosine� hydrogen b.

Tyr&tryp > polar & absrb UV light at 280

Asparagine & Asparagine & glutamine � amide

whereas

Aspartate & glutamate Aspartate & glutamate �COOH

at pH 7.0

Log Io/I=ecl=A

e =molar extinction coefficient (in units of liters per mole-centimeter)

C=concentrationC=concentration

Protrombin: a blood clooting protein

Modification protein

Plant cell wall

collagenElastin:fibrous proteinprotein

Zwitterion (hybrid ion)

• in water, both ions + & - The image part with relationship ID rId4 was not found in the file.& -

• A zwitterion acts as a base and an acid (give base and an acid (give and recieve H) : amphoteric (both amphoteric (both donor and acceptor) often called often called ampholytes.

Diprotic form of glysinDiprotic form of glysin

•Deprotonation of 2 groups:

•First� carboxyl group•First� carboxyl group

•Then� amino group

•pI: isoelectric point•pI: isoelectric point

•net charge is “0”

•Where first proton release •Where first proton release finish, second begin

•pI=(pK1+pK2)=(9,6+2,34)/2=5,97

pKa of COOH of acetic acid = 4.76pKa of COOH of glycine= 2.34Over 100> acidicWHY ;Because in zwitterions, nearby amino gropu affect the deprotonation of WHY ;Because in zwitterions, nearby amino gropu affect the deprotonation of groupEnzymes-specific sites�interaction�morechanges in pKa

3 stages: one for R

so 3 pKa valuesso 3 pKa values

pI calculation is difficult.

Peptides & peptide bonds

Peptides:2-2 or 3 thousands AA

Peptide bond: name of covalent Peptide bond: name of covalent bond (covalently joined through a substituted amide linkage)

Dehydration

nucleophile

Dehydration

2:dipeptide; 3:tripeptide; 4:tetrapeptide; 5:pentapeptidehydration dehydration

>3(a few): oligopeptide

Many a.a.�polypeptide (lower than 10000 Dalton)

dehydration

Dalton)

>10.000 �protein

Pentapeptide:5 peptide bonds

Aminoterminal end�N-terminalAminoterminal end�N-terminal

Carboxyl-terminal end�C-terminal

Hydrolysis of peptide� slowly-exergonic:::::t : 7 yearsHydrolysis of peptide� slowly-exergonic:::::t1/2: 7 years

+1

At pH 7At pH 7

-1-1

Net charge=(+1)+(-1)+(+1)+(-1)=1-1+1-1=0

+1

-1

Activity of proteins

• Not depending on size– Small ones � active– Small ones � active

• Example: aspartame � sweety dipeptide

• Not exactly depending on concentration• Not exactly depending on concentration– Low concentration�active

• Exp:hormone, poison• Exp:hormone, poison– Oxytocin (9aa) � uterine contraction– Bradykinin (9aa)�inhibitor for tissue inflaammation– Tyrotropin-releasing factor (3aa)�from – Tyrotropin-releasing factor (3aa)�from

hypoth.�pitut.glan�thyropin release– Amanitin (mushroom poison) small and effective

Larger proteins

• Such as insulin– 2 polypeptide chain

• 30 aa residues in one• 30 aa residues in one

• 21 other

• Glucagon� 29 aar• Glucagon� 29 aar

How long can a ppc be?

Single poylpeptide chainSingle poylpeptide chain

2 or > polypeptide chain linked NONCOVALENTLY: multisubunit protein.protein.

Hemoglobin: 4 subunit

•Oligomer (protein) : if at least 2 are identical

•identical units �protomers

•Hemoglobin: a tetramer of 4 ppsu

What about insulin?

•Hemoglobin: a tetramer of 4 ppsu or a dimer of alfa-beta protomers

No, it has 2 ppc but linked by disulfide bond.

Average MW of aa

Average MW of 20 aa�138Average MW of 20 aa�138

But smallers are prodominant

So average MW accepted�128

But dehydration means lost water MW of water �18 But dehydration means lost water MW of water �18 (O+2H)

128-18=110128-18=110

AND SO AMW�110

Hydrolyse proteins with acid

But, Asparagine and glutaminebecome aspartate and glutamate

Tryptopan ���� degradedTryptopan ���� degraded

Serine, threonine, tyrosin ����lost

Extra chemicals in proteins

• Simple Proteins– Many � no other, full aa

• Example: ribonuclease A, chymotrypsinogen• Example: ribonuclease A, chymotrypsinogen

• Conjugated Proteins• Conjugated Proteins– some � permenantly associated chemicals to aa.

• These groups� prosthetic groups– Lipoproteins– Lipoproteins

– Glycoproteins

– Metalloproteins

Levels of Protein Structure

PS: a description of Covalent Bonds(PB&diSB) linking aa in a ppc

SS: stable arrangments of aa residues giving rise to recuring SS: stable arrangments of aa residues giving rise to recuring structural patterns

TS: complete 3D folding of a PPCTS: complete 3D folding of a PPC

QS: if 2->subunit, their arrangmets in space

Aim of Protein Purification

- confirm sequence information

- identify sites of post translation modif-ication - identify sites of post translation modif-ication eg. -phosphorylation sites

- glycosylation sites

- lipid attachment sites- lipid attachment sites

- enzyme characterization

- crystal structure - crystal structure

- produce antibodies

PURITY

Selection of Protein Source

• Animal, plant, bacteria (E.coli), fungi (Saccharomyces cerevisia)(Saccharomyces cerevisia)– Easy to optain– Easy to optain

– Amount of the protein in tissue

– Select any properties of the portein peculiar to only it.only it.

Methods of Solubilization

1. Liberate from the cell: Consider1. Liberate from the cell: Consider1. Mechanical feature of the cell1. Mechanical feature of the cell

2. Location of the protein on the cell1. Membrane bound proteins 1. Membrane bound proteins

2. Cytosolic proteins: Break open the cell

1. Osmatic lysis: Hypothonic solution1. Osmatic lysis: Hypothonic solution

2. Lysozyme: enzyem to digest the cell wall of bacteria

3. Detergant or organic solvents (acetone, toluene) to 3. Detergant or organic solvents (acetone, toluene) to break down the cell.

4. Mechanical distruption4. Mechanical distruption» Grinding with sand or alumina

» High speed blender

» Sonicator» Sonicator

» French press

Combine some of them

What will happen to cell lysate

• Centrifugation

• Filtration• Filtration

• Dylasing• Dylasing

Filtering or DialysingFiltering or Dialysing

If the protein is in an organelle of the If the protein is in an organelle of the cell.cell.

• We should remove or seperate that organelles.organelles.

• (Differential) Centrifugation is the best • (Differential) Centrifugation is the best technique

Removal of Non-Protein Removal of Non-Protein ComponentsComponents

Rate-zonal centrifugationRate-zonal centrifugation

Density Gradient Centrifugation

Stabilizing Protein

• Denatured protein is useless

• What effect the structure of protein» pH» pH

» Ionic strength

» Temperature (purification: 0-5oC)

» Protelytic effect (proteases)» Protelytic effect (proteases)

» Some are sensitive to cysteine residues to form disulfide bonds; heavy metal contaminations; salt disulfide bonds; heavy metal contaminations; salt concentration; polarity of solution

• To prevent the growth microorganisms � NaN3

(Sodium azide)3

(Sodium azide)

Some proteins have extrema properties Some proteins have extrema properties that can be useful for purificationthat can be useful for purification

• Proteins resistant to protlytic degradation • Proteins resistant to protlytic degradation

(proteases). Autlysis degrades others

• Proteins.. cold cold-labile or heat-stable.

• Heat the mixture, only heat-stable ones will

stay others aggregate and precipitatestay others aggregate and precipitate

What we should do for stability

• Use– Buffer

– Work at 40C– Work at 4C

– Proteinase inhibatorsInhibitor Enzymes Working concentration

Prot

eol

ytic

inh

ibit

ors Inhibitor Enzymes Working concentration

Diisopropyl fluorophosphate (DFP) Serine proteases Avoid DFP

Phenylmethylsulfonyl fluoride (PMSF) Serine proteases 0.5-1 mM

Prot

eol

ytic

inh

ibit

ors

(PMSF) Serine proteases 0.5-1 mM

EDTA Metalloproteases ~ 5 mM

Iodoacetamide/

iodoacetic acidCysteine proteases 0.1 mM

Prot

eol

ytic

inh

ibit

ors

iodoacetic acid

Pepstatin Aspartic proteases 1 µM

LeupeptinSerine proteases/ cysteine

proteases1 µMPr

oteol

ytic

inh

ibit

ors

Assays for Proteins

• Tracking proteins and our protein is

important during prufication

Protein determination (eg Abs at 280 nm, Bradford

assay, Lowry assay) to track the protein existance.

• Enzyme: If the desired protein is an enzyme, it is

very easy to detecte the protein by tracking an very easy to detecte the protein by tracking an

activity in the solution.

– Coupled enzyme reaction: Sometimes we can’t – Coupled enzyme reaction: Sometimes we can’t

visulize the product of a reaction but convert it to

another enzymatic product to visulize.

• Normal Protein: Using ELISA

General Strategy of P.P.Characteristics ProcedureSolubility 1. Salting in

2. Salting out2. Salting out

Ionic Charge 1. Ion exchange Chr.

2. Electrophoresis

3. Isoelectric focusing3. Isoelectric focusing

Polarity 1. Adsorption C.

2. Paper C.

3. Reverse-phase C.

4. Hydrophobic interaction C.

Molecular Size 1. Dialysis & Ultrafiltration

2. Gel electrophoresis

3. Gel filtration C

4. Ultracentrifugation4. Ultracentrifugation

Binding Specificity 1. Affinity C

Ammonium Sulfate Precipitation

• Relies on fact that proteins loose solubility as concentration of salt is increasedconcentration of salt is increased– Is characteristic of particular protein

– Results in a partial purification of all proteins with – Results in a partial purification of all proteins with similar solubility characteristics

– Must determine [amm sulf] to precipitate your protein – Must determine [amm sulf] to precipitate your protein empirically.

• Produces “salt cuts”• Produces “salt cuts”

Salting in / Salting out

• Salting IN• At low concentrations,

• Salting OUT• At high concentrations • At low concentrations,

added salt usually increases the solubility of charged macromolecules

• At high concentrations added salt lowers the solubility of macromolecules because it charged macromolecules

because the salt screens out charge-charge

macromolecules because it competes for the solvent (H2O) needed to solvate out charge-charge

interactions.• So low [salt] prevents

aggregation and therefore

(H2O) needed to solvate the macromolecules.

• So high [salt] removes the solvation sphere from the aggregation and therefore

precipitation or “crashing.”

solvation sphere from the protein molecules and they come out of solution.“crashing.” they come out of solution.

Kosmotrope vs. Chaotrope

• Ammonium Sulfate• Increasing conc causes

• Urea• Increasing conc • Increasing conc causes

proteins to precipitate stably.

• Increasing conc denatures proteins; when they finally do stably.

• Kosmotropic ion =

when they finally do precipitate, it is random and

• Kosmotropic ion = stabilizing ion. random and

aggregated.

• Chaotropic ion = denaturing ion.denaturing ion.

Fractionation types and ordersFractionation types and orders

• Strategies• Strategies– 1. step: Solubility.

• adjust charge (pH, temperature, [salt])• adjust charge (pH, temperature, [salt])

• ↑ [salt] � less soluble�centrugation�precipation– Mostly used salt� ammonium persulfate (NH) SO )– Mostly used salt� ammonium persulfate (NH4)2SO4)

– Process� salting out

– 2. Dialysis– 2. Dialysis– Both purification and removal of salts

– 3. Chromotography– 3. Chromotography

Chromotographic Seperations

• 1903, Russian botanist M. Tswett����leaf pigments• Chromotography:chroma=color;graphein=write• Chromotography:chroma=color;graphein=write• Two phases: mobile & stationary phases

– Mobile Phase: a mixture is dissolved in MP. MP �gaseous or liquid. liquid.

– Stationary Phase: Porous solid matrix. – Solutes are fractionated according to the types and strength of

interaction between solutes and stationary phase. interaction between solutes and stationary phase. • There are many Modern Chromotographic Methods• We group them according to

– Types of Stationary Phase– Types of Stationary Phase• Gas-liquid chromotography• Liquid-liquid chromotography

– Types of the interaction between SP and solutes– Types of the interaction between SP and solutes• Example: ion exchange chromotography; adsorption chromotography

Protein Purification: Column ChromatographyProtein Purification: Column Chromatography

• The expansion of the protein band in the mobile phase is band in the mobile phase is caused by separation of proteins with different properties and by diffusional spreading. As the length of the column increases, length of the column increases, the resolution of two types of protein improves.

• Rate is decreased and resolution can decline because of the diffusional spreading diffusional spreading

Ion Exchange Chromotography1. IONIC ELUTION2. pH ELUTION

Ion Exchange Chromotography

Low saltP+ + Na+ Na+ + P+

High saltP+ + Na+ Na+ + P+

• Charge of protein

– At pI, it is zero, above pI, negative and below pI, positive. Each and below pI, positive. Each protein has characteristic pI

• Anion exchange

– Use resin that has positive charge. – Use resin that has positive charge. Use a pH above pI of protein. Protein of interest adheres and drive off with salt gradient.drive off with salt gradient.

– Proteins with highest pIs elute first

– DEAE cellulose (or sephadex)

• Cation exchange• Cation exchange

– Use resin that has negative charge and use at pH below pI of proteins.and use at pH below pI of proteins.

– Proteins with highest pIs elute last

– CM-cellulose or Sephadex.

Functional group used in ion exchangers.

Ion exchange groups used in protein purification

STRONG CATION─SO3

- ─ Sulpho

STRONG ANION─ CH2N+(CH3)3 ─ Triethylaminomethyl ─SO3

- ─ Sulpho

─ CH2SO3- ─ Sulphomethyl

─ C3H6SO3- ─ Sulphopropyl

─ CH2N+(CH3)3 ─ Triethylaminomethyl

─ C2H4N+(C2H5)3 ─ Triethylaminoethyl

─ C2H4N+(C2H5)2CH2CH(OH)CH3 ─ Diethyl-2-hydroxy-propylaminoethyl

WEAK CATION ─ COO- ─ Carboxy

─ CH COO- ─ Carboxymethyl

propylaminoethyl

WEAK ANION ─ C2H4N+H3 ─ Aminoethyl

─ C2H4N+H(C2H5)2 ─ Diethylaminoethyl ─ CH2COO- ─ Carboxymethyl ─ C2H4N+H(C2H5)2 ─ Diethylaminoethyl

Summary of IECR+A- +B-� R+B- +A-R+A- +B-� R+B- +A-

R+A-=ion (anion) exchangerB-=anion in the solutionB-=anion in the solutionProteins have both anion & cation. Net Charge � stength of its binding to inert exchangerNet Charge � stength of its binding to inert exchangerBut in solution there is salt SO competition between salt and

protein.protein.pH is very importantTwo thinks improtantTwo thinks improtant1) Salt concentration 2)pHWhy improtant? Strongly binding of protein to IECWhy improtant? Strongly binding of protein to IEC

Some terms:Some terms:

• Elution: a process of motion of proteins with less affinity to the matrix in a buffer with less affinity to the matrix in a buffer with known [salt] and pH value.with known [salt] and pH value.

To elute the protein, use 1)more buffer 2) a different buffer wth different [salt] or/and pH

• Stepwise Elution: an elution of protein • Stepwise Elution: an elution of protein using different buffer with different [salt]

Gel Filtration ChromotographyGel Filtration Chromotography

• Many names• Many names– Gel filtration

– Size exclusion

– Molecular sieve chromotograpy

• How proteins are seperated?• How proteins are seperated?• According to the shape and size

• Mobile phase�buffers;

• stationary phase �beads (SEPHADEX):hydrated, spongelike with pores

• Bigger ones move rapidly

• Smaller ones move slower

Gel filtration• Porous beads made of different

materials. Size of pores can be • Porous beads made of different

materials. Size of pores can be controlled

• Small molecules small enough to go • Small molecules small enough to go into beads whereas larger go around and thus flow faster. There is and thus flow faster. There is exclusion limit (all proteins too large to go into pores).to go into pores).

• Can be used as preparative method or be used to determine molecular sizeor be used to determine molecular size

• Gels made of dextrans, agarose or polyacrylamidepolyacrylamide

• Dialysis uses size difference– Utilize membrane with (typically) 10 kd – Utilize membrane with (typically) 10 kd

cutoff. Method for exchanging salts

Terms used in GFCTerms used in GFC

• Exclusion limit: the MW of smallest molecule can’t penetrate the pores of the beads (shaped can’t penetrate the pores of the beads (shaped affects).affects).

• Molecules <exclusion limit elute according to the • Molecules <exclusion limit elute according to the size. Bigger ones elute first.

• Elution time: time to elute the protein.

• According to 2 plot types, MW determined• According to 2 plot types, MW determined– 1. Elution type to MW plot

– 2. Ve/Vo to MW plot

• Vt=Vx + Vo• Vt=Vx + Vo– Vt: total bed volume– Vo:void volume (volume of solvent space surrounding

beads.beads.– Vx: volume occupied by the gel beads (Vx=Vi (internal

space of beads) + Vgel (volume of solid parts of gel)space of beads) + Vgel (volume of solid parts of gel)» Vo� ~34% of Vt

• Elution volume (Ve): volume of solvent to • Elution volume (Ve): volume of solvent to elute the solute from the column (saturation volume of column with buffer)volume of column with buffer)

• Relative elution volume=Ve/Vo

Vo

VtVo Vt

standarts samplestandarts sample

55

65

45E

lutio

n T

ime

Proein MW Elution Time

10000 30

6000 47 25

35

Elu

tion

Tim

e

3000 60 0 2000 4000 6000 8000 10000 12000

MW50?

5300

5300

Terms used in GFCTerms used in GFC• Exclusion limit: the MW of smallest molecule • Exclusion limit: the MW of smallest molecule

can’t penetrate the pores of the beads (shaped affects).affects).

• Molecules <exclusion limit elute according to the size. Bigger ones elute first.size. Bigger ones elute first.

Ve/

Vo

Advantages of Gel ExclusionAdvantages of Gel Exclusion1. Separations can be done over large range of

pH, T, I, and solventspH, T, I, and solvents

2. Virtually no adsorption or loss of material or 2. Virtually no adsorption or loss of material or denaturation

3. Less zone spreading than with most other 3. Less zone spreading than with most other methodsmethods

4. Elution volume related to M in simple way

ApplicationsApplications1. De-salting – use low MW gel column;

protein in void volume – salt laterprotein in void volume – salt later

2. MW determinations - +/- 10% - protein still 2. MW determinations - +/- 10% - protein still in native form

3. Study binding of small molecules – ligands –3. Study binding of small molecules – ligands –use column equilibrated with small molecule ligand; then put protein through column and ligand; then put protein through column and monitor elution profile – see ligand peak and monitor elution profile – see ligand peak and can measure binding constant

Question1:– An enzyme (MW:24000 & pHI:5,5) is

contaminated with a protein of similar contaminated with a protein of similar molecular weight, but with pHI: 7,0 and another protein (MW:100,000; pHI:5,4)protein (MW:100,000; pHI:5,4)

– Suggest a purification

• Q2:– The protein albumin (pH4,6), urease (pH5,0) – The protein albumin (pHI:4,6), urease (pHI:5,0)

and myoglobin (pHI:7,0) were applied to a column of DEAE-Cellulose at pH 6,5. The column of DEAE-Cellulose at pH 6,5. The column was eluted with a dilute pH 6,5 buffer, and then with the same buffer containing and then with the same buffer containing increasing concentrations of NaCl. In what order will the proteins be eluted from the order will the proteins be eluted from the column?

Affinity ChromotographyAffinity Chromotography

Separation by binding affinity -Separation by binding affinity -Affinity chromatography

• Relies on the ability of a protein to bind specifically Affinity chromatography

• Relies on the ability of a protein to bind specifically

to another molecule.to another molecule.

• Columns are packed with beads with covalently

attached ligand molecules that bind to protein of attached ligand molecules that bind to protein of

interest.

• Elution with excess ligand, salt, pH, denaturation

• Antibody affinity chromatography• Antibody affinity chromatography• Histidine/Nickel columns

Antibody affinity chromatographyAntibody affinity chromatography

Matrix for affinity-IMatrix for affinity-I

• Agarose� the best– Many functional groups

(hydroxyl group) to (hydroxyl group) to immobilize ligand.

• Ligand covalently linked to • Ligand covalently linked to agarose

– Buy reacted agarose with cyanogen bromidecyanogen bromide

– Ligand treated with it to form covalent linkscovalent links

Matrix for affinity-II• But many proteins can’t bind its ligand

because of steric interference of agarose

Matrix for affinity-II

because of steric interference of agarose

• Spacers are used: commercial resins• Spacers are used: commercial resins– Example� epoxy-activated resins

Epoxy group can react with many nucleophilic group on ligand.ligand.

How to elute the protein

1. Use its ligand

Addition of glucose (G)

2. Change salt, pH and temperature of buffer2. Change salt, pH and temperature of buffer

Elution methodMethod 1Method 1The simplest case. A change of buffer composition elutes the bound substance without harming either it or the ligand.

Method 2Extremes of pH or high concentration Extremes of pH or high concentration of chaotropic agents are required for elution, but these may cause permanent or temporary damage.

Method 3 and 4Specific elution by addition of a Specific elution by addition of a substance that compete for binding. These methods can enhance the specificity of media that use group-specific ligands.specific ligands.

• One unit is 1 mmol of product formed per minute.• One unit is 1 mmol of product formed per minute.– if 1 ml of solution having enzyme causes formation of 71.4 mmol of product

formed per minute, • the enzyme stock activity is 71.4 unit per 1 ml.• the enzyme stock activity is 71.4 unit per 1 ml.

• Total Activity (TA) is activity/ml Xtotal volume (ml).– 71.4 X 1400 = 100000 units

• Specific Activity (SA) = activity of 1 mg of protein• Specific Activity (SA) = activity of 1 mg of protein– 100.000/10.000=10 units/mg

• Yield of purification= TA of last step/ TA of previous one – 96000/100000=0.96% yield– 96000/100000=0.96% yield

• Fold purification = SA of last step/ SA of previous one .– 32/10=3.2 folds

Analysis of Proteins

• Electrophoresis (analytic method)– Under electric fields

– Many types– Many types• SDS-PAGE �MW

• Native PAGE � native MW• Native PAGE � native MW

• Isoelectric Focusing Electrophoresis � pI

• Two Dimensional Electrophoresis � mixed• Two Dimensional Electrophoresis � mixed

SDS-PAGE

• Cross-linked polyacrylamide: like molecular sievemolecular sieve

• seperate according to the charge to mass ratio– Shape affects– Shape affects– µ(electrophoretic mobility of a molecule)=V(velocity of a

protein)/E(electric potential)=Z(net charge of molecule)/f(frictional coefficent)molecule)/f(frictional coefficent)

• Treated with SDS – SDS�proteins shape similar– SDS�proteins shape similar

• Proteins become negatively charged

Coomassie blue

if protein � oligomerics => if protein � oligomerics => subunits are seperated by SDS and 2-mercaptoethanol (for and 2-mercaptoethanol (for denaturing) used before electrophoresis

Estimate the unknownEstimate the unknown

- -

-load standards----

--------load standards

++

- Let’s draw Rf and logMW plot

run standard Q1: What is Rf?Q2: How to calculate Rf?run standard proteins and tracking dye We know their MW

A-200000Q1: What is Rf?Q2: How to calculate Rf?

B- 100000The Rf is the ratio of the distance migrated by the molecule to that migrated by a tracking dye

Rf value = [distance of protein migration] / [distance of tracking dye migration]

C- 50000

migrated by a tracking dyemigration]

distance of tracking dye migration = 12.5cmC- 50000 distance of tracking dye migration = 12.5cm

+

D- 20000TrackingDye Tracking dye

+

How to draw Rf and logMW plot

Summarize it

protein

Log (MW) Rf

A 5,30 0,19

B 5,00 0,45

C 4,70 0,72

D 4,30 0,94

How to draw Rf and logMW plot

Log (MW)

Rf (MW)

A 5,30 0,19logMW-Rf PLOT

1,0

B 5,00 0,45 0,8

1,0

Rf

0,3

0,5Rf

C 4,70 0,72 0,0

4,0 4,4 4,8 5,2 5,6

logMW

D 4,30 0,94

-distance of sample protein migration = 7.4 cmdistance of sample protein migration = 7.4 cm

Rf = 7.4 / 12.5 = 0.59

logMW-Rf PLOT

1,3

0,8

1,0

1,3

Rf

0,0

0,3

0,5R

f

y = -0,7599x + 4,2427

R2 = 0,9883

0,0

4,0 4,4 4,8 5,2 5,6

logMW4.8

+ 104.8=63.095+ 104.8=63.095

A-200000

B- 100000

C- 50000

Unknown: 63095

C- 50000

D- 20000TrackingDye

http://www.rit.edu/~pac8612/electro/Electro_Sim.html

Isoelectic Focusing Electrophoresis

• Using ampholytes (low MW acids & bases) � a pH gradient established � a pH gradient established

http://www.shsu.edu/~chm_tgc/sounds/flashfiles/CEs.swf

Two-dimensional ElectrophoresisTwo-dimensional Electrophoresis

Covalent Structure of ProteinsCovalent Structure of Proteins• # and type of aa in each protein�different

• PS�SS�TS�QS�Function– E.coli �3.000 different protein– E.coli �3.000 different protein

– Human � 25.000-30.000 protein

– Primary structure– Primary structure• �function of protein

• �diseases– Small change in PS�lost of function

– But, smtimes nothing

» 20-30% of proteins�polymorhic (varians in aa)» 20-30% of proteins�polymorhic (varians in aa)

• Similar function�highly diffrent funciton

• Some regions are conserved�functionally • Some regions are conserved�functionally important

• (1953) James D. Watson & Francis • (1953) James D. Watson & Francis Crick � double helix structure of DNA• its precise replication• Frederick Sanger �sequence of aminoacid residues of insulin • seen DNA --- protein relation• seen DNA --- protein relation•Decade after, DNA codes protein• DNA database �exponantially growth• DNA database �exponantially growth• protein database�slowly• Estimate protein from DNA• But, still experimental methods being used

Sanger developed many Sanger developed many principles to sequence polypeptidespolypeptides

PP Sequences

• Short PP can be sequenced

• Label aa on N-terminal (Sanger’s Mthd)– Label it wth one of them– Label it wth one of them

– 1-fluoro-2,4-dinitrobenzene (FDNB)

– Dansyl chloride– Dansyl chloride

– Dabsyl chloride

•Label N terminal•Hydrolize it to aa•HPLC•Detect which one it is

Edman Degradation

Pehr EdmanFrom N Terminal, PTC to aa�PTC adduct From N Terminal, PTC to aa�PTC adduct ~~> purify and identifyMachine to sequence PP�SEQUENATOR

problems in sequencinghttp://www.wiley.com/college/fob/quiz/quiz05/5-15.swf

problems in sequencing

Processing row protein

• Denaturation: heat or urea

• Break disulfide bonds: 2-marceptoethanol (performic acid or dithiothreitol-DTT)and (performic acid or dithiothreitol-DTT)and stabilize wth iodoacetate

Cutting polypeptide Cutting polypeptide chainchain

by

• enzymes called

by

• enzymes called proteases proteases (hydrolytic cleavage) cutting cleavage) cutting PP from a specific site.site.

Other techniques for pp Other techniques for pp sequencingsequencing

• Mass spectrometry �short ones (<20 aa) in a few minutes.a few minutes.

• DNA sequencing�protein sequencing• DNA sequencing�protein sequencing

• Proteome�entireprotein complements encoded by genomeencoded by genome

Mass Spectrometry-MALDI-TOF

Peptide Synthesize• Many peptides�Pharmacologic agents• Many peptides�Pharmacologic agents• To obtain them• To obtain them

1. Purify from tissue (low concentration)2. Genetic engineering2. Genetic engineering3. Direct synthsize

– Hard but not impossible– Hard but not impossible– Not whole one, portion of a protein �important– Classic organic chemistry methods�impracticale– Classic organic chemistry methods�impracticale

– Why? Because of prufication in each step

How to modern synthesize

• B. Merrified,1962� novel tech. • Not prufication• Not prufication• Peptide immobilize to a matrix• The matrix�insoluble polypeptide beads• N and C terminus blocked for unwanted • N and C terminus blocked for unwanted

binding

Hero chemical�FMOC Hero chemical�FMOC 9-fluorenylmethoxycarbonyl9-fluorenylmethoxycarbonyl

limitations

•Efficiency of chemicals

•Bad chemical�unfinished reaction� incomplete cycle�impurity (short and cycle�impurity (short and complete peptides all together)together)

•Automated

•100 aa in a few days

Novel peptides can be ligated to other proteins�novel Novel peptides can be ligated to other proteins�novel protein�???

Guess. What is happining

Wild type mutantWild type

3D of proteins

• PS�SS�3D of protein�protein�function�location of proteinprotein

• 3D�important– Still we dont know

how 3D occur

Protein Family

• Protein families, and their related domains, are defined by sequence homology.defined by sequence homology.

• Proteins in a family– 25% identical– 25% identical– Some Common in

• Function• Function• Structure

• BUT difficulties• BUT difficulties– Ex: some proteins in a PF, a few aa common for a

special function

• Certain aa� like signal• Certain aa� like signal

• İf it exist we estimate– Cellular location– Cellular location

– Post-transcriptional modifications– Post-transcriptional modifications

– Half-life

– Used to transport– Used to transport

– Zip protein

– Attachments for prosthetic group (sugar etc)– Attachments for prosthetic group (sugar etc)

Protein AlignmentProtein Alignment

• Pauling and Emile Zuckerkandl �

molecular evolutionmolecular evolution

• The more similarity�the closer relation• The more similarity�the closer relation

• Carl Woese, 1970�rRNA for archea

• Protein sequence�refine, clarify the discus

Changes in Proteins

• Conserved regions � activity for protein• The less important�the more variance• The less important�the more variance• Substitute aa in a sequence• Substitute aa in a sequence

RESİM YAP tolerateRESİM YAP

REZİM YAP

REJİM YAP

Some substitutes are OKEY

tolerate

REJİM YAP But some not

• Lateral gene transfer:• Lateral gene transfer:

A BB

Ex: antibiatic resictance gene

Moleculer evolution�on protein familyMoleculer evolution�on protein family

Proteins in a PF carries key funciton in the metobolism of each. Ex: EF-1α in eukaryotes, EF-Tu in pro.(elongation in each. Ex: EF-1α in eukaryotes, EF-Tu in pro.(elongation in translation)

Members of PF� homologous proteins (homologs)Members of PF� homologous proteins (homologs)

homologs

ParalogsParalogs (same species)

ortologsortologs (different species)

The more The more homologs The more relation

How to measure level of homology

• By computer• By algorithm • By algorithm • Most importance

– Sliding method:– Sliding method:• Slide words• Give score• Give score

– Scoring �highly different from one another

kastamonuastımkastamonuastım

Gap formation� better alighment

Penalties� avoid uninformative penalties

Kasaphayrinerede

ka - -pta----n

Best scoring

• Not yes or no

• Some aa� similar function, structure– Glutamine~asparagine– Glutamine~asparagine

– Alanine~Valine Higher score

Blosum tableBlosum table

This is Blosum62

62% identical proteins of PF with full function compared 62% identical proteins of PF with full function compared and scored.

Blosum50

Protein� more reliable than NA Protein� more reliable than NA sequencesequence

• Exactly

• NA� actac: – 3 codon�act,cta,tac– 3 codon�act,cta,tac

– Add reverse cat, atc, tca

• Protein� one informative result• Protein� one informative result– Lower uninformative aligment chance– Lower uninformative aligment chance

Ef-1α & Ef-Tu

Specific sequence � unique to taxonomic group