Amino Acids, Peptides, and Proteins

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• Part I

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Amino Acids, Peptides, and Proteins

• Amino Acids
• Peptides and Proteins
• Protein Structure
• Protein Sequences

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a-carboxyl group
a-amino group
?-carbon
Side chain
General structure of amino acid
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For all the standard amino acids except
glycine,the a-carbon is covalently bonded to
four different groups (-COO-, -NH3, -H and
-R).The a-carbon is a chiral center.All
molecules with a chiral center are also
optically active.
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• The two stereoisomers of alinine, L- and
  D-alanine, are nonsuperimposable mirror images of
  each other (enantiomers).

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Steric relationship of the stereoisomers of
alanine to the absolute configuration of L- and
D-glyceraldehyde. The amino acid residues in
proteins are L stereoisomers
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Amino acids can be classified by R group
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Tryptophan and tyrosine absorb ultraviolet light.
The absorbance maxima for both occurs near a
wavelength of 280 nm. Phenylalanine generally
contributes little to the absorbance properties
of proteins,
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• Reversible formation of a disulfide bond by the
  oxidation of two molecules of cysteine. Disulfide
  bonds between Cys residues stabilize the
  structures of many proteins

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• The additional carbons in an R group are commonly
  designated ?, ?, ?, ?, and so forth, proceeding
  out from the ? carbon,

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Peptides and Proteins

• The polymers of amino acids that are linked by
  peptide bonds. The protein is a polypeptide.

Formation of a peptide bond by condensation
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The pentapeptide Ser-Gly-Tyr-Ala-Leu
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Biologically active peptides and polypeptides
occur in a vast range of sizes

• Oxytocin nine amino acid residues, secreted by
  the posterior pituitary and stimulates uterine
  contractions.
• Glucagon 29 residues, a pancreatic hormone,
  opposing the action of insulin.

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Lengths of proteins vary considerably
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Different proteins contain different amino acid
compositions
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Some proteins contain chemical groups other than
amino acids
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Levels of structure in proteins
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• Column Chromatography

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3. Ion-exchange Chromatography
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4. Size-exclusion chromatography
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5. Affinity chromatography
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• Electrophoresis a technique for the separation
  of proteins is based on the migration of charged
  proteins in an electric field.
• Electrophoresis of proteins is generally carried
  out in gels made up of the cross-linked polymer
  polyacrylamide.
• The polyacrylamide gel acts as a molecular sieve.
  Separation of proteins is based on their
  charge-to mass ratio.
• Electrophoresis in the presence of SDS separates
  proteins on the basis of molecular weight.

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• SDS binds to most proteins in amount roughly
  proportional to the molecular weight of the
  protein.
• The bound SDS contributes a large net negative
  charge, rendering the intrinsic charge of the
  protein insignificant.
• The native conformation of a protein is altered
  when SDS is bound.

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Different samples are loaded in well at the top
of the polyacrylamide gel.
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• After electrophoresis, the proteins are
  visualized by
• adding a dye such as Coomassie blue, which binds
  to proteins

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• SDS-PAGE can estimate the molecular weight of a
  protein

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• Isoelectric focusing a technique used to
  determine the isoelectric point.
• A pH gradient is established by allowing a
  mixture of ampholytes to distribute themselves in
  an electric field generated across the gel.
• When a protein mixture is applied, each protein
  migrates until it reaches the pH that matches its
  pI.

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• Isoelectric focusing

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Two-dimensional electrophoresis
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• More than 1,000 different proteins from E coli
  can be resolved using this technique

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• The Function of a protein depends on its amino
  acid sequence

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• The bacterium E. coli produces more than 3,000
  different proteins a human produces 25,000 to
  35,000.
• Each type of protein has a unique
  three-dimensional structure and this structure
  confers a unique function.
• Each type of protein has a unique amino acid
  sequence.

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• Thousands of human genetics diseases have been
  traced to the production of defective proteins.
• Perhaps one-third of these proteins are defective
  because of a single change in their amino acid
  sequence hence, if the primary structure is
  altered, the function of the protein may also be
  changed.
• On comparing functionally similar proteins often
  have similar amino acid sequences, as an extreme
  case is ubiquitin.

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Is the amino acid sequence absolutely fixed, or
invariant, for a perticular protein?

• NO some flexibility is possible.
• An estimated 20 to 30 of the proteins in human
  are polymorphic, having amino acid sequence
  variants in the human population.
• Many of these variations in sequence have little
  or no effect on the function of the protein.

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• Although the amino acid sequence in some regions
  of the primary structure might vary considerably
  without affecting biological function, most
  proteins contain crucial regions that are
  essential to their function and whose sequence is
  therefore conserved.

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Methods of protein sequencing

• Automated Edman degradation
• DNA sequencing
• Mass spectrometry

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Amino acid sequence of protein can be deduced by
its DNA sequence
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The Nobel Prize Winners in Chemistry 2002
J. B. Fenn (1917)
K.Tanaka (1959)
Kurt Wüthrich
ESI
SLD
NMR
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Protein Sequences and Evolution

• Protein sequences are a rich source of
  information about protein structure and function,
  as well as the evolution of life on this planet.

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A bacterial evolutional tree, based on the
sequence divergence observed in the GroEL family
of protein