Chapter 3: Amino Acids, Peptides, and Proteins

Proteins
Most aboundant In great variety
Size Charge Function Etc

Informative Genes expressed as protein Monomer aminoacids, joint by a covalent (peptide) bond
20 types
Side chain

Like letters, different combinations different meanings

Hormone, fibers, enzymes, antibodies, transporters, lens proteins, feather, spiders webs, poisins etc First asparagin (1806) in asparagus Glutamate in wheat gluten Tyrosin in chees (greek tyros) Glysin .. (greek sweet glykos)

Firefly light by luciferase, luciferins, ATP

hemoglobins

Keratin : rhinoceros horn, nail etc

Structure of AA
All aa
C linked 3 groups
An amino A carboxyl A R group:side Normally 20 but some more but not as structural, for special function in specific proteins

Assigned for each

3-letter abbreviations 1 letter symbol

Addtion carbon to C or 1.2(C ).3.4.5 (start from highest atomic number)

A leter code devised by Dayhoff

The founder of the field of bioinformatics CysteinHistidineIsoleuniceMethionineSerineValinefir st letter unique AlanineGlycineLeuicneProlineThreoinefirst letter NOT unique but aa more common aRginine,Fenylalanine,tYrosine,tWiptophanphonetical ly suggestive asparDate,asparagiNe,glutamEte,Qtamineletters within their names or suggested by names Lysine (K) K comes after L

 1839 G.J. Mulder used the term protein First aminoacid isolated in 1830; 20the one isolated in 1930 20 standard aminoacid (AA); -amino acids AAdipolar ions, (both ions;zwitterions) Amino groups and carboxyl groupsreadly ionized. pK (pK1) values of carboxyl groups of AA: ~2.2 pK (pK2) values of amino groups of AA: ~7.4 At pH 7.4, amino groupprotonated (+); carboxyl group deprotonated (-) pKRdepends on functional groups

At physiological pH

Zwitterion (hybrid ion)

in water, both ions + & A zwitterion acts as a base and an acid (give and recieve H) : amphoteric (both donor and acceptor) often called ampholytes.

Condensation rxn: bond formation while eliminating water CO-NH: PEPTIDE BOND Aa come togetgerpolymer 2 aadipeptide; 3tripeptide; tetrapeptide = oligopeptide Polypeptide and oligopeptide = peptides Aa in peptides amino acid residues

Peptides & peptide bonds

Peptides:2-2 or 3 thousands AA
nucleophile

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

hydration

dehydration

2:dipeptide; 3:tripeptide; 4:tetrapeptide; 5:pentapeptide

>3(a few): oligopeptide Many a.a.polypeptide (lower than 10000 Dalton) >10.000 protein

Polypeptide chain is linear not branched

Pentapeptide:5 peptide bonds Aminoterminal endN-terminal

Carboxyl-terminal endC-terminal
Variation in length and aa compositiondiversity in shape and biological function of proteins

AA Chains Nonpolar, Polar and Charged

According to side chains; Aas divided into 3 type: 1)With nonpolar R group 2)With polar but uncharged R groups 3)With charged R groups

Nonpolar Amino Acid Side Chain

Nine amino acidsnonpolar side chians
Glycine: smallest aa Alanine Valine Aliphatic amino acids Leucine Isoleucine Methionine: thioester side chain Proline: cylic pyrolidine Phenylalanine: aromatic Tryptophan: araomatic with an indole group

Table 4-1 part 1

Table 4-1 part 2

Figure 4-4

Uncharged Polar AA Side Chain

Six amino acids
Serine: OH group Threonine: OH group Asparagine: Amide bearing side chain Glutamine: Amide bsc Tyrosine: phenolic group Cytosine: thiol group that form a disulfide bond

Figure 4-5

Table 4-1 part 3

Charged AA Side Chain

Five amino acids
Basic AA Side Chains (+ charged)
Lysine: butylammonium side chain Arginine: a guanidino group Histidine: an imidazol group

Acidic AA Side Chains (- charg)

Aspartic acid Glutamic acid This classification can be different in different books.

Table 4-1 part 4

Figure 4-7

pK (pKa) Values of Ionic Group

-amino acids have at least 2 acid-base groups (next slides) -aa with ionizable side chains 3 acid-base gr. At pH : 0 all are protonated At pH: 14 all are deprotonated Acidicseasly deprotonated (almost always -) Basicsalmost always protonated (so almost always +) In different pH, different aas charge can be different becasue of pK(1,2,R) of ionizable groups

Diprotic form of glysin

Deprotonation of 2 groups:

First carboxyl group

Then amino group pI: isoelectric point net charge is 0

Where first proton release finish, second begin

pI=(pK1+pK2)=(9,6+2,34)/2=5,97

pKa of COOH of acetic acid = 4.76 pKa of COOH of glycine= 2.34 Over 100> acidic WHY ;Because in zwitterions, nearby amino gropu affect the deprotonation of group Enzymes-specific sitesinteractionmorechanges in pKa

Polyprotic acid

3 stages: one for R so 3 pKa values

pI calculation is difficult.

Charge of proteins
Peptides characteristics titration curve and pI (isoelectric pH (neutral at this pH)) According to the residues pKa value, it is possible to calculate the charge of peptide at a different pH Protein : + charged at pH < pI and - charged at pH>pI Example : if protein As pI is 7 so pH scale

+ + + ++++
1

neutral
7

--

--

--

--

- - -11

10

+1

At pH 7

-1
Net charge=(+1)+(-1)+(+1)+(-1)=1-1+1-1=0
We assume that the charge of residue is the charge of its HA if pH is lower then pKa-1 and it is that of its A if pH is higher than pKa + 1

+1

-1

ON THE OTHER HAND, 3D OF of a folded polypeptide chain may bring polar side chains and the N- and C-termini close together. The resulting electrostatic interactions between these groups may shift their pK values up to several pH units from the values for the corresponding free amino acids. For this reason, the pI of a polypeptide, which is a function of the pK values of its many ionizable groups, is not easily predicted and is usually determined experimentally. To determine pI of peptides, isoelectric focusing gel electrophoresis is used.

Aminoacid Abbreviations
Shown in previous slides In labs, Asn and Gln hydrolized to Asp & Glu That is why Gln and Glu Glx and Asn and Asp Asx AA has ine suffix when they are free AA has yl suffix when they are residues of polypeptides Example:

This tetrepeptide can be writen as; 1. )alanyltyrosylglycine 2. Ala-Tyr-Asp-Gly 3. AYDG

ATOMS OF AA are named with Greek alphabet ,

- carboxyl group - amino group

Figure 4-8

3D structure of AA
Stereochemistry: arrangment of the molecules constituent atoms in 3D space. Stereoisomers: molecules with the same chemicals bonds but different stereochemistry, different configuration (fixed spatial arrangment of atoms) Configuration 1 . double bonds (geometric) Configuartion 2. chiral centers

 AA are optically active (except Gly)

They rotate the light The direction and angle of rotation can be measured by polarimeter.

 If optically activeasymmetric They are not superimposable on their mirror image

Tetrahedral carbon atoms with 4 different substituentsassymetricoptical active: having assymetric center OR chiral center

Chiral center affect on configurational isomers A carbon wth 4 different substiuents asymetric carbon chiral centers (CC) A molecule wth 1 CC 2 stereoisomers 2n: stereoisomer number n:# of CC Enantiomers: having mirror images; chemically the same , physically different Diastereomers : not having m. i.

Enantiomers

Structure and optical activity

No relationship between structure and the degree or direction to which it rotates the plane of polarized light. For example:
Leucine 10.4o to left Arginine 12.5o to right Enantiomers: rotate with the same degree but reverse direction

Fisher Convention configuration of asymmetric centers

Fisher convention used to describe different forms of chiral molecules. 1891, Emil Fisher proposed that the spetial isomers (stereoisomers) Dglyceraldehyde and L-Glyceraldehyde. L (levorotatory) means light to left D (dextrorotatory) to right Fisher projection:
horizontal towards reader Vertical far from reader

 L-Glyceraldehyde and L-AAthe same relative configuration

All AAL stereochemical configuration L does not mean Levorotatory; many are dextrorotatory Life based on chiral molecules
50% of ChemicalsL whereas 50%D after an ordinary chemical synthesis But in living organisms, all are either L or D For example: all amino acidsL configuration becasue enzymes, receptors etc stereospecific

Biological interactions btwn molecules are stereospecific

Some bacterias cell wall contain D-amino acids WHY? Peptidases (protein eating enzymes) produced by their enemies can NOT digest them. Peptides with D-amino acids not made by RIBOSOMEs In pharmacy, many drugs synthesized as racemic mixture (enantiomers L and D together) and packed together WHY? Because of economy isolation of one from otherexpensive But occosionaly, the inactive stereoisomer can be harmful.

4 different groups (exceptglycine)

C chiral center 2 unique spatial arrangments

2 stereoisomers
Nonsuperimposable mirror Enantiomers Optical active L & D forms All (almost) L

Model molecule

Start with carbonyl carbon, if amino group on left L

hydrophil

hydrophob

Used in the estimation of Protein concentration

The Lambert-Beer Law

Majority of biomolecules absorb light at characteristics wavelength (tryptophane absorb light at 280)

log (Io/I)=ecl=Absorbance(A)
e =molar extinction coefficient (in units of liters per mole-centimeter) C=concentration ; l: length of light absorbing sample (fixed to 1 cm)

Adirectly proportional to concentration

Amino Acids Derivatives

20 aa common and genetic codes to them Many other uncommon components of proteins.
Majority seen after posttranslational modification What modificaiton: addition of simple functional group to R side
Hydroxylation, methylation, acetylation, phosphorylation, carboxylation

Figure 4-14

AA and derivatives as messanger

GABA & Dopamine neurotransmitter Histamine local mediator of allergic rxn Thyroxine iodine-containing thyroid hormone stimulating metabolism

sopeptide bond peptide bond GSH keep the cell against oxidative damage

Page 88

Activity of proteins
Not depending on size
Small ones active
Example: aspartame sweety dipeptide

Not exactly depending on concentration

Low concentrationactive
Exp:hormone, poison
Oxytocin (9aa) uterine contraction Bradykinin (9aa)inhibitor for tissue inflaammation Tyrotropin-releasing factor (3aa)from hypoth.pitut.glanthyropin release Amanitin (mushroom poison) small and effective

Larger proteins
Such as insulin
2 polypeptide chain
30 aa residues in one 21 other

Glucagon 29 aar

How long can a ppc be?

Single poylpeptide chain

2 or > polypeptide chain linked NONCOVALENTLY: multisubunit protein.

Hemoglobin: 4 subunit Oligomer (protein) : if at least 2 are identical identical units protomers Hemoglobin: a tetramer of 4 ppsu or a dimer of alfa-beta protomers

What about insulin?

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

Average MW of aa
Average MW of 20 aa138 But smallers are prodominant

So average MW accepted128
But dehydration means lost water MW of water 18 (O+2H)

128-18=110
AND SO AMW110

Hydrolyse proteins with acid But, Asparagine and glutamine become aspartate and glutamate Tryptopan degraded Serine, threonine, tyrosinlost

Extra chemicals in proteins

Simple Proteins
Many no other, full aa
Example: ribonuclease A, chymotrypsinogen

Conjugated Proteins
some permenantly associated chemicals to aa.
These groups prosthetic groups
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 structural patterns TS: 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 eg. -phosphorylation sites - glycosylation sites - lipid attachment sites - enzyme characterization - crystal structure - produce antibodies

PURITY

Selection of Protein Source

Animal, plant, bacteria (E.coli), fungi (Saccharomyces cerevisia)
Easy to optain Amount of the protein in tissue Select any properties of the portein peculiar to only it.

Methods of Solubilization

1. Liberate from the cell: Consider

1. Mechanical feature of the cell 2. Location of the protein on the cell
1. Membrane bound proteins 2. Cytosolic proteins: Break open the cell 1. Osmatic lysis: Hypothonic solution 2. Lysozyme: enzyem to digest the cell wall of bacteria 3. Detergant or organic solvents (acetone, toluene) to break down the cell. 4. Mechanical distruption
Grinding with sand or alumina High speed blender Sonicator French press

What will happen to cell lysate

(crude extract: disrupted cell)
Methods for purification
Chromotography Salting out Centrifugation Filtration Dylasing

The extract is subjected to treatments that seperate the proteins into different fractions based on a property such as size or charge. (fraction is a portion of a mixture that has been subjected to a procedure) Fractionation: the process of seperating the proteins of protein mixture into fractions

Centrifugation:to remove unwanted materials

Removal of Non-Protein Components

Rate-zonal centrifugation

If the protein is in an organelle of the cell.

We should remove or seperate that organelles. (Differential) Centrifugation is the best technique

Density Gradient Centrifugation

Salting Out: Ammonium Sulfate Precipitation

Relies on fact that proteins loose solubility as concentration of salt is increased
Is characteristic of particular protein Results in a partial purification of all proteins with similar solubility characteristics Must determine [amm sulf] to precipitate your protein empirically.

Produces salt cuts

Salting in / Salting out

Salting IN At low concentrations, added salt usually increases the solubility of charged macromolecules because the salt screens out charge-charge interactions. So low [salt] prevents aggregation and therefore precipitation or crashing. Salting OUT At high concentrations added salt lowers the solubility of macromolecules because it competes for the solvent (H2O) needed to solvate the macromolecules. So high [salt] removes the solvation sphere from the protein molecules and they come out of solution.

Kosmotrope vs. Chaotrope

Ammonium Sulfate Increasing conc causes proteins to precipitate stably. Kosmotropic ion = stabilizing ion.
Urea Increasing conc denatures proteins; when they finally do precipitate, it is random and aggregated. Chaotropic ion = denaturing ion.

Filtering or Dialysing
Three missions: 1) Remove excess salt
proteins 3) remove excess water
2) remove unwanted

To remove excess water

To remove excess water and other unwanteds

Centrfigue and small particules will be collected in the tube

Hypotonic solution and remove ions especially after salting out

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 activity in the solution.
Coupled enzyme reaction: Sometimes we cant

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
Solubility

Procedure
1. Salting in
2. Salting out

Ionic Charge

1. Ion exchange Chr.

2. Electrophoresis
3. 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. Ultracentrifugation Binding Specificity 1. Affinity C

Fractionation types and orders

Strategies
1. step: Solubility.
adjust charge (pH, temperature, [salt]) [salt] less solublecentrugationprecipation
Mostly used salt ammonium persulfate (NH4)2SO4) Process salting out

2. Dialysis
Both purification and removal of salts

3. Chromotography

Chromotographic Seperations
1903, Russian botanist M. Tswettleaf pigments
Chromotography:chroma=color;graphein=write Two phases: mobile & stationary phases Mobile Phase: a mixture is dissolved in MP. MP gaseous or liquid. Stationary Phase: Porous solid matrix. Solutes are fractionated according to the types and strength of interaction between solutes and stationary phase.

There are many Modern Chromotographic Methods We group them according to

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

Types of the interaction between SP and solutes

Example: ion exchange chromotography; adsorption chromotography

Protein Purification: Column Chromatography

The expansion of the protein 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, the resolution of two types of protein improves.

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

Ion Exchange Chromotography

1. IONIC ELUTION 2. pH ELUTION

Low salt
P+ + Na+ High salt

Na+ + P+

 Charge of protein At pI, it is zero, above pI, negative and below pI, positive. Each protein has characteristic pI Anion exchange Use resin that has positive charge. Use a pH above pI of protein. Protein of interest adheres and drive off with salt gradient. Proteins with highest pIs elute first DEAE cellulose (or sephadex) Cation exchange Use resin that has negative charge 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 CH2SO3 Sulphomethyl C3H6SO3 Sulphopropyl

COO CH2COO-

WEAK CATION Carboxy

Carboxymethyl

STRONG ANION CH2N+(CH3)3 Triethylaminomethyl C2H4N+(C2H5)3 Triethylaminoethyl C2H4N+(C2H5)2CH2CH(OH)CH3 Diethyl-2-hydroxypropylaminoethyl WEAK ANION C2H4N+H3 Aminoethyl C2H4N+H(C2H5)2 Diethylaminoethyl

Summary of IEC
R+A- +B- R+B- +AR+A-=ion (anion) exchanger B-=anion in the solution
Proteins have both anion & cation. Net Charge stength of its binding to inert exchanger But in solution there is salt SO competition between salt and protein. pH is very important Two thinks improtant 1) Salt concentration 2)pH Why improtant? Strongly binding of protein to IEC

Gel Filtration Chromotography

Many names
Gel filtration Size exclusion Molecular sieve chromotograpy

How proteins are seperated?

According to the shape and size Mobile phasebuffers; stationary phase beads (SEPHADEX):hydrated, spongelike with pores Bigger ones move rapidly Smaller ones move slower

 Porous beads made of different materials. Size of pores can be controlled Small molecules small enough to go into beads whereas larger go around and thus flow faster. There is exclusion limit (all proteins too large to go into pores). Can be used as preparative method or be used to determine molecular size Gels made of dextrans, agarose or polyacrylamide Dialysis uses size difference
Utilize membrane with (typically) 10 kd cutoff. Method for exchanging salts

Gel filtration

Advantages of Gel Exclusion

1. Separations can be done over large range of pH, T, I, and solvents 2. Virtually no adsorption or loss of material or denaturation 3. Less zone spreading than with most other methods 4. Elution volume related to M in simple way

Applications
1. De-salting use low MW gel column; protein in void volume salt later 2. MW determinations - +/- 10% - protein still in native form 3. Study binding of small molecules ligands use column equilibrated with small molecule ligand; then put protein through column 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 molecular weight, but with pHI: 7,0 and another protein (MW:100,000; pHI:5,4) Suggest a purification

 Q2:
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 was eluted with a dilute pH 6,5 buffer, and then with the same buffer containing increasing concentrations of NaCl. In what order will the proteins be eluted from the column?

Affinity Chromotography

 Relies on the ability of a protein to bind specifically to another molecule. Columns are packed with beads with covalently attached ligand molecules that bind to protein of interest. Elution with excess ligand, salt, pH, denaturation

Separation by binding affinity Affinity chromatography

Antibody affinity chromatography Histidine/Nickel columns

Antibody affinity chromatography

HPLC
HPLC: High-performance liquid Chromotography
High pressure pomps to increase the speed

Possitive sites:
Speed t limits diffusional spreading or proteins bands better resolution

Negative site: $

Terms
Unit: 1.0 unit of enzyme activity causes a mol substrate transformation in a minute at room temperature. Activity total activity (as units) of the enzyme solution Specific activity: activity of 1 mg of protein Specific activity = total activity / total mg of protein

 One unit : 1 mol of substrate catalyzed per minute.

the enzyme stock activity is 71.4 unit per 1 ml.

if 1 ml of solution having enzyme causes formation of 71.4 mol of product formed per minute,
71.4 X 1400 = 100000 units

Total Activity (TA) is activity/ml X total volume (ml). Specific Activity (SA) = activity of 1 mg of protein
100.000/10.000=10 units/mg

Analysis of Proteins
Electrophoresis (analytic method)
Under electric fields Many types
SDS-PAGE MW Native PAGE native MW Isoelectric Focusing Electrophoresis pI Two Dimensional Electrophoresis mixed Electrophoresis: migration of molecules in an electric field

SDS-PAGE
Cross-linked polyacrylamide gel: like molecular sieve
seperate according to the charge to mass ratio
Shape affects (electrophoretic mobility of a molecule)=V(velocity of a protein)/E(electric potential)=Z(net charge of molecule)/f(frictional coefficent)

Treated with SDS

SDSproteins shape similar
Proteins become negatively charged

Coomassie blue

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

Estimate MW of the unknown protein

- ---- -

load standards

Lets draw Rf and logMW plot Q2: How to calculate Rf? Q1: What is Rf? run standard proteins and We know their tracking dye MW
The Rf is the = [distance of protein Rf value ratio of the distance migrated by the [distance of tracking migration] / molecule to that migrated by a tracking dye

A-200000

B- 100000

dye

migration]

C- 50000

distance of tracking dye migration= 12.5cm

D- 20000
TrackingDye

Tracking dye

How to draw Rf and logMW plot

Summarize it
protei n A B C D Log (MW) 5,30 5,00 4,70 4,30 Rf 0,19 0,45 0,72 0,94

How to draw Rf and logMW plot

Log (MW) A 5,30 Rf 0,19

logMW-Rf PLOT
1,0 0,8

5,00

0,45

Rf
C 4,70 0,72

0,5 0,3 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 cm Rf = 7.4 / 12.5 = 0.59

logMW-Rf PLOT
1,3 1,0 0,8

Rf
0,5 0,3 0,0 4,0 4,4

4.8 logMW
4,8

5,2 y = -0,7599x + 4,2427 R2 = 0,9883

5,6

104.8=63.095

A-200000

B- 100000

Unknown: 63095

C- 50000

D- 20000
TrackingDye

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

Isoelectic Focusing Electrophoresis

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

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

Two-dimensional Electrophoresis

Covalent Structure of Proteins

# and type of aa in each proteindifferent PSSSTSQSFunction
E.coli 3.000 different protein Human 25.000-30.000 protein Primary structure
function of protein diseases
Small change in PSlost of function

But, smtimes nothing 20-30% of proteinspolymorhic (varians in aa)

Similar functionhighly diffrent funciton Some regions are conservedfunctionally important

 (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 Decade after, DNA codes protein DNA database exponantially growth protein databaseslowly Estimate protein from DNA But, still experimental methods being used

Sanger developed many principles to sequence polypeptides

PP Sequences
Short PP can be sequenced Label aa on N-terminal (Sangers Mthd)
Label it wth one of them
1-fluoro-2,4-dinitrobenzene (FDNB) Dansyl chloride Dabsyl chloride

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

Edman Degradation

Pehr Edman From N Terminal, PTC to aaPTC adduct ~~> purify and identify Machine to sequence PPSEQUENATOR problems in sequencing
http://www.wiley.com/college/fob/quiz/quiz05/5-15.swf

Processing row protein

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

Cutting polypeptide chain by

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

Other techniques for pp sequencing

Mass spectrometry short ones (<20 aa) in a few minutes. DNA sequencingprotein sequencing

Proteomeentire protein complements encoded by genome

Mass Spectrometry-MALDI-TOF

Peptide Synthesize
Many peptidesPharmacologic agents To obtain them
1. Purify from tissue (low concentration) 2. Genetic engineering 3. Direct synthsize
Hard but not impossible Not whole one, portion of a protein important Classic organic chemistry methodsimpracticale
Why? Because of prufication in each step

How to modern synthesize

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

Hero chemicalFMOC 9-fluorenylmethoxycarbonyl

limitations
Efficiency of chemicals Bad chemicalunfinished reaction incomplete cycleimpurity (short and complete peptides all together)

Automated
100 aa in a few days Novel peptides can be ligated to other proteinsnovel protein???

3D of proteins
PSSS3D of protein functionlocation of protein 3Dimportant
Still we dont know how 3D occur

Guess. What is happining

mutant

Wild type

Protein Family
Protein families, and their related domains, are defined by sequence homology. Proteins in a family
25% identical Some Common in
Function Structure

BUT difficulties
Ex: some proteins in a PF, a few aa common for a special function

 Certain aa like signal f it exist we estimate

Cellular location Post-transcriptional modifications Half-life Used to transport Zip protein Attachments for prosthetic group (sugar etc)

Protein Alignment
Pauling and Emile Zuckerkandl molecular evolution The more similaritythe closer relation Carl Woese, 1970rRNA for archea Protein sequencerefine, clarify the discus

Changes in Proteins
Conserved regions activity for protein The less importantthe more variance Substitute aa in a sequence
RESM YAP REZM YAP tolerate Some substitutes are OKEY

REJM YAP

But some not

 Lateral gene transfer:

A
Ex: antibiatic resictance gene

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

Members of PF homologous proteins (homologs)

homologs
Paralogs (same species)
ortologs (different species)

The more homologs

The more relation

How to measure level of homology

By computer By algorithm Most importance
Sliding method:
Slide words Give score

Scoring highly different from one another

kastamonu astm

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 Alanine~Valine
Higher score

Blosum table

This is Blosum62

62% identical proteins of PF with full function compared and scored. Blosum50

Protein more reliable than NA sequence

Exactly NA actac:
3 codonact,cta,tac Add reverse cat, atc, tca

Protein one informative result

Lower uninformative aligment chance

Ef-1 & Ef-Tu

Specific sequence unique to taxonomic group

Page 82

Figure 4-9

Figure 4-10

Figure 4-11

Page 84

Box 4-2

Figure 4-12

Figure 4-13

Figure 4-14

Box 4-3 figure 1

Box 4-3 figure 2

Figure 4-15