Gülüzar_Özbolat_ Aminoacid, Peptides, Protein

7/31/2019 Glzar_zbolat_ Aminoacid, Peptides, Protein

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Amino Acids, Peptides,

Proteins

Structure and naming of amino acids

Structure and properties of peptides

Ionization behavior of amino acids and peptides

Purification and assay methods

Peptide sequencing and chemical synthesis

Protein sequence analysis


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Proteins: Main Agents ofBiological Function

Catalysis:enolase (in the glycolytic pathway)

DNA polymerase (in DNA replication)

Transport:hemoglobin (transports O2 in the blood)

lactose permease (transports lactose across the cell membrane)

Structure:collagen (connective tissue)

keratin (hair, nails, feathers, horns)

Motion:myosin (muscle tissue)

actin (muscle tissue, cell motility)


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Amino Acids: Building Blocks of

Protein

Proteins are heteropolymers of -amino acids

Amino acids have properties that are well

suited to carry out a variety of biologicalfunctions:

Capacity to polymerize

Useful acid-base properties

Varied physical properties

Varied chemical functionality


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Amino Acids: Atom Naming

Organic nomenclature: start from one end

Biochemical designation: start from

-carbon and go down the R-group


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Most -Amino Acids are Chiral The -carbon has always

four substituents and is

tetrahedral

All (except proline) have an

acidic carboxyl group, abasic amino group, and an

alpha hydrogen connected

to the -carbon

Each amino acid has anunique fourth substituent R

In glycine, the fourth

substituent is also hydrogen


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Amino Acids: Classification

Common amino acids can be placed in fivebasic groups depending on their R substituents:

Nonpolar, aliphatic (7)

Aromatic (3)

Polar, uncharged (5)

Positively charged (3)

Negatively charged (2)


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Aliphatic Amino Acids

http://en.wikipedia.org/wiki/File:Aa.svg
http://en.wikipedia.org/wiki/File:Aa.svghttp://en.wikipedia.org/wiki/File:Aa.svg


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Aromatic Amino Acids



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Charged Amino Acids



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Polar Amino Acids



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Special Amino Acids



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Not incorporated by ribosomes

Arise by post-translational modificationsofproteins

Reversible modifications, esp.phosphorylation is important in regulation

and signaling

Uncommon Amino

Acids in Proteins


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The Genetic Code is organized

by Amino Acid Properties


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Ionization

At acidic pH, the carboxylgroup is protonatedand the amino acid isin the cationic form

At neutral pH, the

carboxyl group isdeprotonated but theamino group isprotonated. The netcharge is zero; suchions are called

ZwitterionsAt alkaline pH, the amino

group is neutralNH2and the amino acid isin the anionic form.


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Substituent effects on pKa Values-carboxy group is much more acidic than in carboxylic acids-amino group is slightly less basic than in amines


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Amino Acids CanAct as Buffers

Amino acids withuncharged side-chains,such as glycine, have twopKa values:

The pKa of the -carboxylgroup is 2.34

The pKa of the -aminogroup is 9.6

It can act as a buffer intwo pH regimes.


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Amino Acids Carry a Net Charge

of Zero at a Specific pH

Zwitterions predominate at pH values between the pKavalues of amino and carboxyl group

For amino acid without ionizable side chains, the IsoelectricPoint (equivalence point, pI) is

At this point, the net charge is zero

AA is least soluble in water

AA does not migrate in electric field

2

21pKpK

pI


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How to Calculate the pI When the

Side-chain is Ionizable?

Identify species that carries

a net zero charge

Identify pKa value that

defines the acid strength of

this zwitterion: (pK2)

Identify pKa value that

defines the base strength of

this zwitterion: (pKR)

Take the average of these

two pKa values


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Peptides and Peptide bonds

Peptide bond ina di-peptide

Peptides are

smallcondensationproducts ofamino acids

They are smallcompared toproteins (di, tri,tetra oligo-)


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Peptide Ends are Not the Same

Numbering starts from the amino terminusAA1 AA2 AA3 AA4 AA5


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The Three Letter Code

Naming starts fromthe N-terminus

Sequence is written

as:Ala-Glu-Gly-Lys

Sometimes the one-letter code is used:

AEGK


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Peptides: A Variety of Functions

Hormones and pheromones: insulin (think sugar) oxytocin (think childbirth)

sex-peptide (think fruit fly mating)

Neuropeptides substance P (pain mediator)

Antibiotics: polymyxin B (for Gram - bacteria)

bacitracin (for Gram + bacteria)

Protection, e.g. toxins amanitin (mushrooms)

conotoxin (cone snails)

chlorotoxin (scorpions)


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Proteins are:

Cofactor is a general term for functional non-amino acid component Metal ions or organic molecules

Coenzyme is used to designate an organic cofactors

NAD+ in lactate dehydrogenase

Prosthetic groups are covalently attached cofactors

Heme in myoglobin

Polypeptides (covalently linked -amino acids) + possibly

cofactors,

coenzymes,

prosthetic groups,

other modifications


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Polypeptide Size in Some Proteins


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Classes of Conjugated Proteins

Peptides and Proteins


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Peptides and Proteins-Burning Questions

Sequence and composition?

Three-dimensional structure?

Folding Mechanism?

Biochemical role?

Functional regulation?

Molecular interactions with small and macro-molecules?

Structural and sequence relatives?

Cellular and sub-cellular localization?

Physical and chemical properties?

P ifi ti F ti ti f


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Purification Fractionation ofProtein Mixtures

Separation relies on differences in physico-chemical properties Solubility Selective Precipitation (Centrifugation)

Thermal stability --

Charge --Electrophoresis, Isoelectric Focusing, IEC

Size Dialysis, Sedimentation (Centrifugation), GFC

Affinity for a ligandPull down assays (Centrifugation),AC

Hydrophobicity (HIC)

Chromatography is commonly used for

preparative separation


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Protein Fractionation


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Separation by Charge

Ion Exchange Chromatography

Anion exchange

Matrix positive

Proteins negative

Displaced by anions

Cation exchange Opposite

pH determines net charge on

Proteins

Salt concentration gradient

Native gel electrophoresis

Iso-electric Focusing


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Separation bySize

Size exclusion (Gel

Filtration)

Chromatography Loading vol.


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Separation byAffinity

Affinity

Chromatography

Free Ligand-Beads --centrifugation

Ligand-Magnetic-

Beads

Immuno-assays on

solid supports

El t h i f P t i


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Electrophoresis for ProteinAnalysis

Separation inanalytical scale iscommonly done by

electrophoresis Electric field pulls

proteins according to

their charge Gel matrix hinders

mobility of proteinsaccording to their

size and shape


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SDS PAGE: Molecular Weight

SDS sodium dodecylsulfate a detergent

SDS micelles binds to,and unfold all theproteins

SDS gives all proteins anuniformly negativecharge

The native shape ofproteins does not matter

Rate of movement willonly depend on size:small proteins will move

faster

-


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ProteinSequencing

S i D i f A i


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Spectroscopic Detection of AromaticAmino Acids

The aromatic amino acidsabsorb light in the UV

region

Proteins typically have

UV absorbance maximaaround 275-280 nm

Tryptophan and tyrosine

are the strongest

chromophores Concentration can be

determined by UV-visible

spectrophotometry using

Beers law: A = cl


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Chapter 3: Summary

In this chapter, we learned about:

The many biological functions of peptides and

proteins The structures and names of amino acids found in

proteins

The ionization properties of amino acids and

peptides

The methods for separation and analysis ofproteins


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Nonpolar, Aliphatic R Groups


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Aromatic R Groups

Also

Hydrophobic

These aminoacid side

chains absorb

UV light at270-280 nm


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Polar,

Uncharged RGroups

These amino

acids side

chains can

form

hydrogen

bonding

Cysteine can

form disulfide

bonds


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Basic RGroups


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Acidic R Groups

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Gülüzar_Özbolat_ Aminoacid, Peptides, Protein