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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
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Aromatic Amino Acids
http://en.wikipedia.org/wiki/File:Aa.svg
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Charged Amino Acids
http://en.wikipedia.org/wiki/File:Aa.svg
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Polar Amino Acids
http://en.wikipedia.org/wiki/File:Aa.svg
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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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https://reader009.{domain}/reader009/html5/0501/5ae76969ce298/5ae76986ee935.jpg
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