chapter 3 amino acids and protiens

8/3/2019 Chapter 3 Amino Acids and Protiens

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Amino Acids and the PrimaryStructures of Proteins

Chapter 3

Functions of Proteins Proteins perform many different functions in

the body.

TABLE 19.1


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Amino Acids

Amino acids arecompounds that containboth an amino group anda carboxyl group

-amino acid: an aminoacid in which the aminogroup is on the carbonadjacent to the carboxyl

group All proteins are composed

of 20 standard aminoacids


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Has charged NH3+ and COO- groups. Forms when both the NH2 and the COOH groups

in an amino acid ionize in water. Has equal + and charges at the isoelectric point (pI).

O O +

NH2CH2COH H3NCH2CO

Glycine Zwitterion of glycine

Zwitterions

Fischer Projections of Amino Acids

Are chiral except for glycine. Have Fischer projections that are stereoisomers. L are naturally occurring and are used in proteins.

L-alanine D-alanine L-cysteine D-cysteine

CH2SH

H2N H

COOH

CH2SH

H NH2

COOH

CH3

H NH2

COOH

CH3

H2N H

COOH


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Classification of Amino acids

The R group, or side chain, determines the structuralrange and general physical characteristics of theamino acids

The amino acids are generally grouped according tothe various characteristics of their R groups Non-Polar, Aliphatic R Groups Aromatic R Groups Sulfur-Containing R Groups Side Chains with Alcohol Groups Basic R Groups Acidic R Groups Amides


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Aliphatic R Group


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Aromatic R Group

Spectroscopic Properties

All amino acids absorb in infrared region Only Phe, Tyr, and Trp absorb UV Absorbance at 280 nm is a good diagnostic

device for proteins


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Sulfur-Containing R Group


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Side Chains with Alcohol Groups

Basic R Groups


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Acidic R Groups

Hydrophobicity of Amino Acid SideChains

Hydropathy: the relativehydrophobicity orhydrophilicity of each aminoacid

The larger the hydropathy,the greater the tendency ofan amino acid to prefer a

hydrophobic environment Hydropathy affects protein

folding: Hydrophobic side chains

tend to be in the interior ofa protein and hydrophilicresidiues tend to be on thesurface


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Biologically Active Amino Acids

Amino acids and their derivatives sometimesfunction in non-protein roles in cells.

The most common example are someneurotransmitters and chemical messengers

Non-standard Amino Acids

Non-standard amino acid can be important components ofproteins and peptides

The non-standard amino acids generally results from a specificmodification of an amino acid residue afterit is incorporatedinto the polypeptide strand


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Ionizable Groups in Amino Acids

Amino Acids have acid/ base properties Amino acids are zwitterionic All amino acids have at least two acid base groups

-carboxyl group (pKa 1.8-2.5); unprotonated at pH 7- -amino group (pKa 8.7-10.7); protonated at pH 7

Those with ionizable side chains (R groups) havethree Lysine - Arginine- Glutamate - Aspartate

Histidine - Cysteine Tyrosine

Structure varies with pH

When the pH of the solution is below the pKa of theionizable group, the protonated form of that grouppredominates Acid; proton donor; cation

When the pH of the solution is above the pKa of theionizable group, the unprotonated form of that grouppredominates Conjugate base; proton acceptor; anion


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Amino Acid Titration Curves

Provides quantitativemeasure of the pKa ofeach ionizing group

Provides informationabout the amino acidsbuffering regions

Illustrates the

relationship betweenthe amino acids netelectric charge and thepH of the solution

Has charged NH3+ and COO- groups. Forms when both the NH2 and the COOH groups

in an amino acid ionize in water. Has equal + and charges at the isoelectric point (pI).

O O + NH2CH2COH H3NCH2CO

Glycine Zwitterion of glycine

Zwitterions and Isoelectric Points


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In solutions more acidic than the pI, The COO in the amino acid accepts a proton.

+ H+ +H3NCH2COO H3NCH2COOH

Zwitterion Positive ion

at pI pH< pICharge: 0 Charge: 1+

Amino Acids as Bases

In solutions more basic than the pI, The NH3+ in the amino acid donates a proton.

+ OH

H3NCH2COO H2NCH2COO

Zwitterion Negative ionat pI pH > pICharge: 0 Charge: 1

Amino Acids as Acids


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pH and Ionization

H+ OH

+ +

H3NCH2 COOH H3NCH2COO H2NCH2COO

positive ion zwitterion negative ion

(at low pH) (at pI) (at high pH)

How to calculate pI

The isoelectric point(pI) of an amino acidor peptide is the pH atwhich the charge ofthe molecule = 0.

It can be calculatedsimply as the arithmeticmean of the 2 pKa'scorresponding to thetransitions generatingthe +1 and -1 forms.


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How to calculate pI

Heres how to do it:1. Identify all ionizable groups2. Assign pKas to each ionizable group3. Start with each ionizable group in protonated form

(very low pH maybe 0 or 1) and calculate its netcharge

4. Slowly move up in pH to the first ionizable groupspKa and deprotonate it (reduce charge by 1)

How to calculate pI

5. Do this until each group is deprotonated.Now you have identified all charged formsand at which pH each transition occurs.

6. Identify the form with net charge = 0

7. Take the pKa on either side of the electricallyneutral form and take their average. This isthe pI.


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How to calculate pI

Take Glycine as an example it has only 2ionizable groups. The transition (from lowto high pH) would be:

Gly+1 Gly0 Gly -1

pKa (-CO2H) = 2.4; pKa (-NH3+

) = 9.8pI =(2.4 + 9.8)/2 = 12.2/2 = 6.1


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Isoelectric Point

-Amino Group-CarboxylGroup

Non-polar

pK2pK1Type of AA

How to calculate pI

Glutamate has an ionizable group that generates anegative charge when deprotonated. Its transitionswould be:

Glu+1 Glu0 Glu-1 Glu-2

The relevant pKa's are:pKa (-CO2H) = ?; pKa (R) = ?


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How to calculate pI

Glutamate has an ionizable group that generates anegative charge when deprotonated. Its transitionswould be:

Glu+1 Glu0 Glu-1 Glu-2

The relevant pKa's are:

pKa (-CO2H) = 2.1; pKa (R) = 4.1

pI =(2.1 + 4.1)/2 = 6.2/2 = 3.1

Isoelectric Point

Side Chain-CarboxylGroup

Polar Neg


Non-polar

pK2pK1Type of AA


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How to calculate pI

Histidine has an ionizable group that ispositively charged when protonated. Itstransitions would be:

His+2 His+1 His0 His-1

The relevant pKa 's are

pKa (R) = 6.0; pKa (-NH3+) = 9.3pI =(6.00 + 9.17)/2 = 15.3/2 = 7.65


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Isoelectric Point

-Amino GroupSide ChainPolar Pos

Side Chain-CarboxylGroup

Polar Neg


Non-polar

pK2pK1Type of AA

Peptides and Peptide Bonds

The linear sequence of amino acids in a polypeptidechain is called the primary structure

Amino acids polymerize to form long chains called"peptides"


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The Peptide Bond Is an amide bond. Forms between the carboxyl group of one amino acid

and the amino group of the next amino acid.

O CH3 O+ || + | ||

H3NCH2CO + H3NCHCO

O H CH3 O

+ || | | ||H3NCH2CNCHCO + H2Opeptide bond

Peptides

Individual amino acidsare called amino acidresidues once they areincorporated into apeptide

Polypeptides chains aredescribed by starting atthe N-terminus andproceeding to the C-terminus


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Naming peptides

A peptide is named with A -yl ending for the N-terminal amino acid. The full amino acid name of the free carboxyl group

(COO-) at the C-terminal end.

Both the standard three-letter abbreviations and one-letter abbreviations for the amino acids are used todescribe the sequence of amino acid residues inpeptides and polypeptides.

Glycylglycylalanine Gly-Gly-Ala GGA

Naming peptides


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Peptides and Ionization

Free -amino, -carboxyl group, and Rgroups contribute toacid-base behavior ofpeptides

Protein Purification

In order to characterize a protein fully, it isnecessary to purify it.

Tissue disrupt crude fractionation selected fractionation


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Modulating Solubility

Salting out

At very highconcentrations (>1 M) ofcertain salts, proteinssolubility is reduced due toa competition with the

protein for interaction withwater molecules.

Dialysis

The protein is put in a tube ofcellophane tubing having smallpores of controlled size. Proteins bigger than the poresare retained, while smallermolecules may diffuse out. As the volume of the buffersurrounding the bag is many times(100-1000x) the volume within thebag, the smaller molecules can beeffectively removed after severalchanges of the outer buffer.


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Column Chromatography

Most powerful method for fractionatingproteins

Based on differences in: Protein charge Size Binding affinity


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03-column_chromatography.mov

Ion exchange chromatography

Cation exchangers bearnegatively charged groups.

Anion exchangers bearpositively charged groups.

Polyelectrolytes, such asproteins, can bind to eithercation or anion exchangers,depending on their netcharge (i.e. depending on thepH).


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Gel filtration chromatography(a.k.a. "molecular sieve", "size exclusion")

This technique separatesproteins on the basis ofmolecular size.

The gel is a matrixconsisting of porous beads

The larger proteins will beexcluded from the beadsand will flow through thecolumn faster than thesmaller molecules, whichexperience a much largervolume.

Affinity Chromatography

This technique takesadvantage of the factthat many proteinsspecifically bind othermolecules as part oftheir function. One can

use this information toconstruct a columncontaining the ligandcovalently attached to amatrix.


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HPLC

High Performance Liquid Chromatography Use high-pressure pumps Limits diffusional spreading of protein bands

which improves resolution

Preparative vs. Analytical

Preparative methods used topurifyproteins Able to handle large amounts of protein at once The chromatographic techniques just discussedare all preparative. Analytical methods used to analyze proteins

Usually deal with small amounts of protein Some preparative techniques can also haveanalytical formats.


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Analytical techniques

Separation based on Mass Charge Shape Different combinations of the above

Gel electrophoresis

Separates proteins basedon their migration in anelectric field

Allows for determination ofprotein's isoelectric pointand approximate molecularweight

Typically this is performed inthe presence of a gelsupport, such aspolyacrylamide enhances separation by

serving as a molecularsieve


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Polyacrylamide Gel Electrophoresis(PAGE)

polymerized acrylamidematrix of controlled poresize

buffered to an alkaline pHso most proteins areanionic and migratetoward the anode.

an electric field is applied allows separation of

proteins based on massand charge

SDS-polyacrylamide gel electrophoresis(SDS-PAGE)

Most common form of PAGE Proteins are bound with the

detergent SDS(sodium dodecyl sulfate)

binds proteins at a ratio of ~1SDS per 2 amino acidresidues

protein has a negative chargeroughly proportional to itsmass

The additional negativecharge is much greater thanthe protein's native charge,which can usually beignored.


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SDS-polyacrylamide gel electrophoresis(SDS-PAGE)

As charge/mass ratio isalmost constant and themolecular shapes are allsimilar, separation is on thebasis of mass (molecularweight).

Smaller polypeptides migratefaster and larger onesmigrate slower, due to thegel filtration effect.

There is an empiricalrelationship betweenmobility and molecularweight:

= 1/log Mr

SDS-PAGE Simulation

03_SDS_gel_electro.swf


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Analytical Methods to Analyze Proteins

Methods to determine amino acid

composition

Cleavage of proteins

Protein sequencing


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Hydrolysis of polypeptides& amino acid analysis

Polypeptides can behydrolyzed toconstituent aminoacids.

This is typically done byboilingthe polypeptidein 6 M HCl for 24 hours.

The R groups remain

intact, except for: Trp indole ring

damaged Asn, Gln converted to

Asp, Glu

Amino Acid Analysis

Treatment of proteinhydrolysate withphenylisothiocyanate (PITC)at pH 9.0 to yield PTC-amino acid derivative

Separate by HPLC viahydrophobic attraction of

amino acid side chains tohydrocarbon matrix ofcolumn

Determine concentration bymeasuring absorbance at254nm (due to PTC moiety).


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Amino Acid treated with PITC

Protein Sequencing Strategy: ShortPolypeptides

Sanger Method Label and identify the

amino-terminal residueusing 1-fluoro-2,4-dinitrobenzene (FDNB)yellow derivative

Hydrolyze polypeptideto its constituent aminoacids (using 6 M HCl)

Identify the labeledamino acid bychromatography


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Protein Sequencing Strategy: ShortPolypeptides

Edman Method React peptide with

phenylisothiocyanate whichconverts amino-terminalresidue to aphenylthiocarbamoyl (PTC)adduct.

Cleave peptide usingtrifluoroacetic acid

Convert derivatized amino acidto phenylthiohydantoin

derivative using an aqueousacid Identify the labeled amino acid

Protein Sequencing Strategy for LongPolypeptides

1) Purify protein (methodsdiscussed previously)

2) Cleave disulfides react with:A) reducing agentfollowed by acetylating

agent1) dithiothreitol(DTT)

2) iodoacetate(IAA)

B) performic acid


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3) Cleave polypeptide intosmaller peptidesA) Proteases1) trypsin cleavesafter Lys and Arg2) chymotrypsin cleaves after

Phe, Tyr, or Trp3) endoproteinaseGlu-C cleavesafter Glu


3) Cleave polypeptide into smaller peptidesB) Chemicals

1) Cyanogen Bromide cleaves after Met


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4) Sequence by Edmanprocedure

5) Reassemble sequencethrough overlaps ofpeptides created bydifferent means

6) Map disulfides bycleaving protein intopeptides before disulfidebond cleavage.

chapter 3 amino acids and protiens

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