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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
-Amino Group-CarboxylGroup
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
-Amino Group-CarboxylGroup
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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Protein Sequencing Strategy for LongPolypeptides
3) Cleave polypeptide intosmaller peptidesA) Proteases1) trypsin cleavesafter Lys and Arg2) chymotrypsin cleaves after
Phe, Tyr, or Trp3) endoproteinaseGlu-C cleavesafter Glu
Protein Sequencing Strategy for LongPolypeptides
3) Cleave polypeptide into smaller peptidesB) Chemicals
1) Cyanogen Bromide cleaves after Met
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Protein Sequencing Strategy for LongPolypeptides
4) Sequence by Edmanprocedure
5) Reassemble sequencethrough overlaps ofpeptides created bydifferent means
6) Map disulfides bycleaving protein intopeptides before disulfidebond cleavage.