Chapter Outline

1
Chapter Outline

• Amino Acids
• Amino acid classes Stereoisomers
• Bioactive AA Titration of AA
• Modified AA AA reactions
• Peptides
• Proteins
• Protein structure
• Fibrous proteins
• Globular proteins

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5.1 Amino Acid Definition

• An alpha amino acid is a carboxylic acid with an
  amino group on the carbon alpha to the carboxylic
  acid .
• The alpha carbon also has an R group side chain
  except for glycine which has two Hs.

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Definition, cont.

• If the R group is not H, the AA can exist in two
  enantiomeric forms (nonsuperimposable mirror
  image) forms.)

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Amino Acids

• General form 1. an amino acid (AA) 2. two AA
  linked to form the peptide bond.

L-form
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Amino Acids-2

• Only the L form of amino acids is commonly found
  in proteins.
• Depending on the nature of the R group, AA are
  classified into four groups.
• nonpolar
• polar
• acidic
• basic

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AA with nonpolar side chains-1
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AA with nonpolar side chains-2
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AA with polar side chains-1
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AA with polar side chains-2
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AA acidic and basic
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Amino Acid Titration

• At physiological pH, the carboxyl group of the AA
  is negatively charged and the amino group is
  positively charged.
• Amino acids without charged side chains are
  zwitterions and have no net charge.
  H3N-CHR-COO-.
• A titration curve shows how the amine and
  carboxyl groups react with hydrogen ion.

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Amino Acid Titration-2

• At low pH a nonacidic/nonbasic amino acid is
  protonated and has the structure below.
• H3NCHRCOOH
• The charge behavior of acidic and basic AAs is
  more complex.

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Titration of Alanine
1
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Isoelectric point

• The isoelectric point (pI) for an AA occurs when
  there is no net charge.
• For a neutral AA, the pI is calculated using the
  equation pK1 pK2/2
• Eg. alanine 2.34 9.7 / 2 6.0
• For acidic or basic AAs, the pI is the average of
  the two pKa values bracketing the isoelectric
  structure.

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Isoelectric point-2

• In general the pI is the average of the two pKas
  bracketing the isoelectric structure. Eg.
  glutamic acid, pI 3.2

pK39.9
pK24.3
pK12.2
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5.2 Peptides

• Peptide a polymer of about 2-100 AAs linked by
  the peptide(amide) bond. As the amino group and
  the carboxyl group link, water is lost.

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Peptides-2

• A peptide is written with the N-terminal end to
  the left and the C-terminal end to the right.
• H2N-Tyr-Ala-Cys-Gly-COOH
• Name Tyrosylalanylcysteinylglycine
• The peptide bond is rigid and planar due to the
  resonance contribution shown right.

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Peptides-3

• The peptide bond angles force specific
  conformations of proteins and, on extended
  chains, successive R groups are on opposite sides.

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Physiologically Interesting Peptides
Common name carnosine found in muscle tissue
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Physiologically Interesting Peptides
Glutathione the reduced form reduces oxidizing
agents by dimerizing to form the disulfide bond
with release of 2 H.
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Physiologically Interesting Peptides
Tyr-Gly-Gly-Phe-Leu
C-terminal AA
N-terminal AA
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Physiologically Interesting Peptides
Oxytocin Induces labor and aids in forcing
milk from the mammary glands.
Vassopressin has a Phe at position 3 instead of
Ile and an Arg at position 8 instead of a Leu.
Its role is in regulating blood pressure.
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Protein Function

• Catalysis
• 2. Structure
• 3. Movement
• 4. Defense
• 5. Regulation
• 6. Transport
• 7. Storage
• 8. Stress Response

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Proteins by Shape-1

• Fibrous proteins exist as long stranded
  molecules Eg. Silk, collagen, wool. A collagen
  segment in space-filling mode illustrates this
  point.

Red spheres represent oxygen, grey carbon, and
blue nitrogen
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Proteins by Shape-2

• Globular proteins have somewhat spherical shapes.
  Most enzymes are globular. Eg. myoglobin,
  hemoglobin. Myoglobin in space-filling mode is
  the chosen example.

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Proteins by Composition

• Simple
• Contain only amino acids
• Conjugated
• simple protein (apoprotein)
• prostetic group (nonprotein)
• glycoproteins
• lipoproteins
• metaloproteins
• etc.

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Four Levels of Protein Structure

• Primary, 1o
• the amino acid sequence
• Secondary, 2o
• 3-D arrangement of backbone atoms in space
• Tertiary, 3o
• 3-D arrangement of all the atoms in space
• Quaternary, 4o
• 3-D arrangement of subunit chains

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Determining Primary Structure

• 1. Hydrolyze protein with hot 6M HCl.
• Identify AA and of each.
• Usually done by chromatography
• 2. Identify the N-term and C-term AAs
• C-term via carboxypeptidase
• N-term via Sangers Reagent, DNFB
• 2,4-dinitrofluorobenzene
• Often step 2 can be skipped today.

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Det. Primary Structure 2

• 3. Selectively fragment large proteins into
  smaller ones.
• Eg. Tripsin cleave to leave Arg or Lys as C-term
  AA
• Eg. Chymotrypsin cleave to leave Tyr or Trp or
  Phe as C-term AA
• Eg. Cyanogen bromide cleaves at internal Met
  leaving Met as C-term homoserine lactone

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Det. Primary Structure 3

• 4. Determine AA sequence of peptides with AA
  sequencer using Edmans reagent
• phenyl isothiocyanate which reacts with the
  N-term AA
• See the next slide

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Det. Primary Structure 3b
protein
Edmans reagent
Phenylthiohydantoin (PTH) derivative of N-term AA
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Det. Primary Structure 4

• 5. Reassemble peptide fragments from step 3 to
  give protein.
• An example follows on the next slide.

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Det. Primary Structure 4b

• A twelve AA peptide was hydrolyzed.
• Trypsin hydrolysis
• Leu-Ser-Tyr-Gly-Ile-Arg
• Thr-Ala-Met-Phe-Val-Lys
• Chymotrypsin hydrolysis
• Val-Lys-Leu-Ser-Tyr
• Gly-Ile-Arg
• Thr-Ala-Met-Phe
• Deduce the AA sequence

Lys is internal!
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Det. Primary Structure 4c
Keeping in mind the N-term AA and overlaping the
sequences properly gives

• Tr
  Leu-Ser-Tyr-Gly-Ile-Arg
• Ct
  Gly-Ile-Arg
• Ct
  Val-Lys-Leu-Ser-Tyr
• Tr Thr-Ala-Met-Phe-Val-Lys
• Ct Thr-Ala-Met-Phe
• The complete sequence is
• Thr-Ala-Met-Phe-Val-Lys-Leu-Ser-Tyr-Gly-Ile-Ar
  g

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Secondary Structure

• The two very important secondary structures of
  proteins are
• a-helix
• b-pleated sheet
• Both depend on hydrogen bonding between the amide
  H and the carbonyl O further down the chain or on
  a parallel chain.

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a Helix Peptide w Hbonds
First six CO to N hydrogen bonds shown
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b Sheet stick form Protein G
H bonds in dotted red-blue
Chain segment 1
Seg 2
Seg 3
Chain 1
Seg 4
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B Sheet Lewis Structure
Antiparallel sheet
Parallel sheet
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Supersecondary Structure

• Reverse turns in a protein chain allow helices
  and sheets to align side-by-side
• Common AA found at turns are
• glycine small size allows a turn
• proline geometry favors a turn

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Supersecondary Structure 2
Combinations of a helix and b sheet.
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Tertiary Structure

• The configuration of all the atoms in the protein
  chain
• side chains
• prosthetic groups
• helical and pleated sheet regions

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Tertiary Structure 2

• Protein folding attractions
• 1. Noncovalent forces
• a. Inter and intrachain H bonding
• b. Hydrophobic interactions
• c. Electrostatic attractions
• to - ionic attraction
• d. Complexation with metal ions
• e. Ion-dipole
• 2. Covalent disulfide bridges

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Tertiary interactions diag.
metal coordn
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Domains

• Domains are common structural units within the
  protein that bind an ion or small molecule.

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Quaternary Structure-1

• Quaternary structure is the result of noncovalent
  interactions between two or more protein chains.
• Oligomers are multisubunit proteins with all or
  some identical subunits.
• The subunits are called protomers.
• two subunits are called dimers
• four subunits are called tetramers

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Quaternary Structure-2

• If a change in structure on one chain causes
  changes in structure at another site, the protein
  is said to be allosteric.
• Many enzymes exhibit allosteric control features.
• Hemoglobin is a classic example of an allosteric
  protein.

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Denaturation

• -loss of protein structure, 2o? 4o, but not 1o.
• 1. Strong acid or base
• 2. Organic solvents
• 3. Detergents
• 4. Reducing agents
• 5. Salt concentrations
• 6. Heavy metal ions
• 7. Temperature changes
• 8. Mechanical stress

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Denaturation-2

• Denaturing destroys the physiological function of
  the protein.
• Function may be restored if the correct
  conditions for the protein function are restored.
• But! Cooling a hardboiled egg does not restore
  protein function!!

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Fibrous Proteins

• Fibrous proteins have a high concentration of
  a-helix or b-sheet. Most are structural
  proteins.
• Examples include
• a-keratin
• collagen
• silk fibroin

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Globular Proteins

• Usually bind substrates within a hydrophobic
  cleft in the structure.
• Myoglobin and hemoglobin are typical examples of
  globular proteins.
• Both are hemoproteins and each is involved in
  oxygen metabolism.

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Myoglobin 2o and 3o aspects

• Globular myoglobin has 153 AA arranged in eight
  a-helical regions labeled A-H.
• The prosthetic heme group is necessary for its
  function, oxygen storage in mammalian muscle
  tissue.
• His E7 and F8 are important for locating the heme
  group within the protein and for binding oxygen.
• A representation of myoglobin follows with the
  helical regions shown as ribbons.

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Myoglobin 2o and 3o aspects
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The Heme Group
N of His F8 binds to fifth site on the iron.
His E7 acts as a gate for oxygen.
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Binding Site for Heme

• Lower His bonds covalently to iron(II)
• Oxygen coordinates to sixth site on iron and the
  upper His acts as a gate for the oxygen.

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Hemoglobin

• A tetrameric protein
• two a-chains (141 AA)
• two b-chains (146 AA)
• four heme units, one in each chain
• Oxygen binds to heme in hemoglobin cooperatively
  as one O2 is bound, it becomes easier for the
  next to bind.
• Lengthy segments of the a and b chains homologous
  to myoglobin.

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Hemoglobin ribbons hemes

• Each chain is in ribbon form and color coded.
• The heme groups are in space filling form

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Oxygen Binding Curves

• Oxygen bonds differently to hemoglobin and
  myoglobin.
• Myoglobin shows normal behavior while hemoglobin
  shows cooperative behavior. Each oxygen added to
  a heme makes addition of the next one easier.
• The myoglobin curve is hyperbolic.
• The hemoglobin curve is sigmoidal.

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Oxygen Binding Curves-2
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The Bohr Effect (H and Hb)

• Lungs
• pH higher than in actively metabolizing tissue.
  (Low H). Hb binds oxygen and releases H.
• Muscle at Work
• pH lower (H product of metabolism). Hb releases
  oxygen and binds H.