Amino Acids, Peptides, and Proteins

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Chapter 3

• Amino Acids, Peptides, and Proteins

"You do not really understand something unless
you can explain it to your grandmother."     
Albert Einstein.
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General Structural Features of Amino Acids
All naturally-occurring amino acids are found as
L stereoisomers. (Remember Fisher Projections
Carbon in most oxidized state is on top chiral
carbon is at center amino group is on LEFT for
the L isomer.)
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General Structural Features of Amino Acids
All naturally-occurring amino acids are found as
L stereoisomers. (Remember Fisher Projections
Carbon in most oxidized state is on top chiral
carbon is at center amino group is on LEFT for
the L isomer.)
The amide bond
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Beers Law
T I/I0 Abs -log T
Abs ?bc where ? is extinction coefficient, b
is path length, and c is molar concentration
QUESTION What are the units of ??
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Amino acids have acid-base properties
Zwitterionic form
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• Isoelectic point (pI) pH at which net charge 0
• pI ½ (pK1 pK2) (for non-ionizable R groups)
• Found at inflection point of titration curve

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Non-polar amino acids
Glycine (Gly (G))
Alanine (Ala (A))
Valine (Val (V))
Leucine (Leu (L))
Methionine (Met (M))
Proline (Pro (P))
Isoleucine (Ile (I))
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Aromatic Amino Acids
Phenylalanine (Phe (F))
Tyrosine (Tyr (Y))
Tryptophan (Trp (W))
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Polar amino acids
Serine (Ser (S))
Threonine (Thr (T))
Glutamine (Gln (Q))
Cysteine (Cys (C))
Asparagine (Asn (N))
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Cationic Amino Acids
Histidine (His (H))
Lysine (Lys (K))
Arginine (Arg (R))
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Anionic Amino Acids
Aspartate (Aspartic acid) (Asp (D))
Glutamate (Glutamic acid) (Glu (E))
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Ionization of a tetrapeptide
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Peptide Bond Formation(Dehydration reaction)
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Levels of Protein Structure

• Primary (1) Covalent sequence of amino acids
• Secondary (2) Local 3-dimensional structure
  forces/bonds are mainly non- covalent
• Tertiary (3) Long range, full 3-dimensional
  conformation
• Quaternary (4) Arrangement in space of
  multi-subunits

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Protein Separation and Purification

• Fractionation Usually salted out, using
  solubility to separate
• Ionic strength ? I 1/2?ciZi2
• Dialysis Separation of proteins (large
  molecules) from other, smaller ones using a
  semi-permeable membrane

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Protein Separation and Purification

• Fractionation Usually salted out, using
  solubility to separate
• Ionic strength ? I 1/2?ciZi2
• Dialysis Separation of proteins (large
  molecules) from other, smaller ones using a
  semi-permeable membrane
• Chromatography Separation by size, charge,
  affinity

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Column Chromatography

• Glass column is filled with resin, a porous
  material (stationary phase)
• Protein-containing mixture is placed on top of
  column, which migrates through column (mobile
  phase)
• Fractions (effluent) are collected in tubes from
  bottom of column
• Fractions are analyzed using various methods

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Ion-exchange chromatography

• Proteins are separated by CHARGE
• Resin contains covalently-attached charged
  molecules
• Movement of proteins with opposite net charge
  elute slower than those with like charges
• pH of buffer can be adjusted as needed rate of
  elution is determined by net charge at given pH

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Size-exclusion chromatography

• Proteins are separated by SIZE
• Resin contains specifically sized PORES
• Large proteins elute FIRST because smaller ones
  are trapped in the pores

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Affinity chromatography

• Proteins are separated by degree of interaction
  with specific molecules
• Specific molecules are covalently attached to
  resin
• Proteins with higher AFFINITY to such molecules
  elute slower than all others
• Very powerful method antibodies and enzyme
  substrates are often used because of the very
  strong interactions and specificity

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Protein AnalysisElectrophoresis

• Proteins migrate by electrostatics according to
  size and shape through polyacrylamide gel (rate
  of migration a z/m)
• E potential
• v velocity of migration
• Z net charge on protein
• f frictional coefficient
• ? electrophoretic mobility
• ? v/E Z/f

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To separate proteins exclusively by size

• SDS-PAGE (Sodium dodecyl sulfate)
• Detergent denatures protein
• Negative charge neutralizes charge on protein
• Stoichiometry is ? 1 SDS/2 amino acid residues,
  i.e., binding is proportional to Mr of protein

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Isoelectric Focusing, 2-D Electrophoresis

• Create pH gradient proteins migrate on gel to
  the position of their pI
• Proteins are resolved by pI (1st dimension) and
  Mr (2nd dimension)

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PurificationHow do we know?

• For enzymes
• Activity 1.0 unit of activity ? quantity of
  enzyme needed to turn over 1.0 ?mol
  substrate/min at 25 C under optimal conditions
• pH, concentrations, etc.
• Specific activity ? quantity of units/mg total
  protein
• For other proteins
• Binding assays
• Secondary effects (e.g., signaling)

Each step of purification is assayed
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Protein Primary Structure

• Linear sequence of amino acids determines
  secondary, tertiary, and quaternary structures
• Primary sequence can be determined indirectly
  from genes or directly from protein
• Disulfide bonds are the other covalent bond
  involved in many proteins
• Several chemical methods are commonly used for
  protein sequencing

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Amino termini
Bovine insulin (Frederick Sanger)
Disulfide bonds
Carboxyl termini
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Amino Terminus Labeling
Dansyl chloride
Dabsyl chloride
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Edman Degradation
Edmans Reagent
Peptide is derivatized by reagent at amino
terminus work-up results in labeled residue and
peptide with n-1 amino acids. Reaction is
repeated
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New slide! (Figure 3-26)
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Sequencing of proteins can be achieved by
protease digestion

• Hydrolyze and identify amino acids
• Identify N-terminus (FDNB)
• Digest with proteases
• Sequence small peptide chains (Edman degradation)
• Locate/quantify disulfide bonds
• See example in Figure 3-27

Newer and faster methods exist, including DNA
sequencing and subsequent analysis using the
Genetic Code (more to come on this)
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Peptides can be chemically synthesized

• Merrifield solid support Protected amino
  acids are covalently attached to resin
• Protecting group is removed
• Next protected amino acid is added
• Etc.