Chapter 5 Amino Acids, Peptides, and Proteins

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Chapter 5 Amino Acids, Peptides, and Proteins

• Polymeric macromolecules are highly ordered.
• Sequence Structure and Function
• Covalent and non-covalent interactions

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Amino Acids, Peptides, and Proteins

• Proteins
• Wide range of sizes within cell
• Diversity of Function
• Constructed from 20 naturally occurring Aas (and
  some non-natural AAs)

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Protein Structure and Function

• Proteins dehydration polymers of AAs (show
  reaction)
• AAs Arginine (1806) Threonine (1938)
• All are a-amino acids
• Additional carbons in R group identified using
  Greek letters
• All are chiral except glycine

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Ch. 5 (Contd)

• Enantiomers identified using D, L System
  (Relative system based on Glyceraldehyde)
• AAs in proteins are exclusively L (D located in
  bacterial cell walls)
• Asymmetric nature of enzyme (AA synthesis)

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Classification of AAs By Polarity of R Group
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Classification (Contd)

• Nonpolar, Aliphatic
• Aromatic R Groups
• Polar, Uncharged R Groups
• Positively Charged (Basic) R Groups
• Negatively Charged (Acidic) R Groups
• Review Structures of AAs

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Nonstandard Amino Acids

• 4-hydroxyproline
• 5-hydroxylysine
• 6-N-methyllysine
• Selenocysteine
• 300 additional amino acids have been found in
  cells.

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Titration Curves

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Titration (Contd)

• Glycine pKa COOH 2.34
• pKa CH3COOH 4.8
• Isoelectric Point
• pI ½(pK1 pK2)

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Peptides and Proteins

• Peptide linkage (bond) bond between two amino
  acid residues
• Equilibrium favors reactants not products
• OH as the leaving group
• Oligopeptide several AAs linked together
• Polypeptide - gt10 AAs bonded together

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Polypeptides

• N (amino) terminal
• C (carboxy) terminal
• Hydrolysis of peptide linkage is exothermic
• occurs slowly due to high activation energy
• stable half life of 7 years under most
  intracellular conditions

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Titration of Polypeptides

• Only one a-amino and a-carboxy group
• Characteristic titration curves like free Aas as
  well as a pI.

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Size of Proteins

• L-Aspartyl-L-phenylalanine methyl ester
  (Aspartane)
• Oxytocin (9 AAs) stimulates uterine
  contractions
• Bradykinin (9 AAs) inhibits inflammation of
  tissues
• Thyrotropin-releasing factor (3 AAs) stimulates
  release of thyrotropin
• Titin (27,000 AAs) constituent of vertebrate
  muscle

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Proteins Involving More than 1 PP Chain

• Multisubunit proteins with two or more PPs
  associated noncovalently
• maybe identical or different
• If at least two are identical, oligomeric
  identical units referred to as protomers
• E.g. Hemoglobin 2 identical a chains and two
  identical ß chains

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PPs Have Characteristic AA Sequences

• Conjugated proteins contain permanently
  associated chemical components non-AA part
  called a prosthetic group.
• lipoproteins, glycoproteins, metalloproteins

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Four Levels of Protein Structure

• Primary (1o) AA sequence
• Secondary (2o) stable arrangements giving rise
  to recurring structural patterns
• Tertiary (3o) 3-D folding
• Quatenary (4o) relationship between individual
  PP chains

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Protein Purification

• Column Chromatography takes advantage of
  differences in protein charge, size, binding
  affinity, etc.
• A. Cation Exchange (Neg. charged solid matrix)
• HPLC polarity
• Electrophoresis migration of charged proteins
• Isoelectric Focusing determine pI of protein,
  pH gradient established using known acids and
  bases

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Covalent Structure of Proteins

• Different AA sequence Different Function
• 1/3 of known genetic diseases result from change
  on a single AA
• Ubiquitin (76 Residues) protein degradation
• AA sequence the same in fruit fly and humans
• 20-30 of proteins in humans are polymorphic (AA
  sequence variance in the human population)

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A Bit of History

• 1953 Watson and Crick discover structure of DNA
• 1953 Fredrick Sanger works out sequence of the
  hormone insulin
• Use of Human Insulin (Recombinant DNA Technology)
  v. Bovine Insulin (Hypoglycemic Awareness)
• insulin level high v. blood sugar level
  (hunger, sweating, and poor coordination)

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History (Contd)

• Sickle Cell Anemia replacement of Glu at
  position 6 of beta chain with Val (146 residues)
• hydroxyurea stimulates bone marrow to
  synthesize fetal hemoglobin which do not have
  beta chains

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Determination of AA Sequence

• 1960s role of DNA nucleotide sequence in
  determining AA sequence revealed.
• Short Automated Sequencers (hydrolysis)
• N-terminus 2,4-DNP, Phenylisothiocyanate
• number of polypeptides

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Edman Degradation

• Remove N-terminus AA
• New N-terminus label and removed and so on
• Determined with a few micrograms of a protein

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Determination of Sequence of Larger proteins

• Accuracy decreases as protein gets larger.
• 1. Cleavage of Disulfide Bonds
• a. Oxidation by performic acid
• b. Reduction by Dithiothreitol (followed by
  acetylation by iodoacetate)
• 2. Fragmenting the PP chain using proteases
  (cleave peptide bond adjacent to specific AA).

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Proteases

• Trypsin Lys, Arg (C)
• Submaxillarus protease Arg (C)
• Chymotrypsin Phe, Trp, Tyr (C)
• Staphylococcus aureus (V8 protease) Asp, Glu
  (C)
• Pepsin Phe, Trp, Tyr (N)
• Cyanogen bromide Met (C)
• Work sample problem in book.

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Synthetic Peptides and Proteins

• Three Methods of Obtaining
• Isolation from Tissue (low conc.)
• Genetic Engineering
• Direct Chemical Synthesis

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Peptide Synthesis

• Merrifield (1962) peptide synthesis while
  anchoring one end to a solid support (insoluble
  polymer resin)
• Synthesis involves building up peptide one AA at
  a time.
• Use of protecting groups to prevent side
  reactions.

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Synthesis (Contd)