Protein folding

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Protein folding

• Protein secondary structure

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Determining Primary Structure

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

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Acid-catalyzed hydrolysis of a peptide
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Sequence determination

• Dansyl chloride method
• Edman degradation (N-terminal sequencing)
• Proteolytic enzymes (cleavage at specific sites)
• Cyanogen bromide (methionine residues)

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Dansyl chloride reaction
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Edman degradation
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Non-enzymatic cleavage (Cyanogen bromide CNBr)
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Protease enzymes
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Cleavage and sequencing
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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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Rotations

• Rotations can take place about the N-Ca (f) and
  about the Ca-CO (?)
• The closer ? and f are to 180o or -180o, the more
  extended (ß-sheet) overall structure is
• The closer ? and f approach zero, the more
  compact (a-helix) and coiled overall structure is

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Alpha helix and Beta sheet

• Most frequently encountered regular secondary
  structures of polypeptides
• In a -helix
• Hydrogen bonds are within single polypeptide
• In ß-sheet
• Hydrogen bonds are between adjacent chains of
  polypeptides

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ß-Pleated sheet

• The residues are more stretched out (flat ribbon)
• Each residue is rotated 180o with respect to
  preceding residue
• Carbonyl oxygen and amido groups point in
  opposite directions
• Hydrogen-Bonding is between neighboring chains
• Can either be parallel or anti-parallel (N-C or
  C-N)

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• Helices may be either right handed (clockwise) or
  left handed (anti-clockwise), viewed from the Ca
• The a-helices in proteins are all right handed
• a-Helical content of proteins ranges from none
  to almost 100

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Terminology

• Axis central line of symmetry running through
  the helix
• Pitch (p) spacing distance between individual
  adjacent coils of the helix
• Repeat (c) distance along the axis it takes for
  a helix to exactly repeat itself
• Rise (h) distance the helix rises between
  adjacent residues

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• Consider a-helix as a spiral staircase
• The rise (h) is the height of each step
• The pitch (p) is the distance from where one is
  standing to the corresponding spot directly above

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• For a-helix, it takes exactly 18 amino acids to
  repeat itself
• Amounts to 5 turns (for repeat)
• Has 3.6 residues per turn
• Rise is 0.15 nm per residue
• Pitch is therefore 0.54 nm

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Rules governing folding

• Minimal Bond length and angle distortion
• Atom proximity must be within allowable van der
  Waals radii
• Amide must be planar and in trans configuration
• Non-covalent bonding stabilizes folding

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• The carbonyl oxygen is hydrogen bonded to amido
  (from amide) hydrogen on the fourth residue up
  the helix
• There are 13 atoms from the H-bonded carbonyl
  group to the amido hydrogen
• Results in linear Hydrogen bonding

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