The primary sequence of proteins

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The primary sequence of proteins Chapters 3 and 4

• Levels of protein structure
• Determining the sequence of amino acids in a
  polypeptide
• chain.
• A. Sequencing the protein.
• B. Sequencing the DNA.
• What determines the sequence of amino acids in a
• polypeptide chain?
• 4. Calculating the charge on a peptide.

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• Levels of protein structure
• A. Primary structure.
• Sequence of amino acids in a protein.
• Covalent bonds peptide bonds.
• B. Secondary structure local hydrogen bonds
• a-helix
• b-sheet
• C. Tertiary structure general fold nonpolar
  interactions.
• D. Involves gt 1 polypeptide chain.

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What is a polypeptide chain? Linear sequence of
amino acids held together by covalent (peptide)
bonds. Proteins contain one or more polypeptide
chains.
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• Determining the sequence of amino acids in
• a polypeptide chain.
• Sequencing the protein
• 1) The Edman degradation.

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First, reduce the disulfide bonds.
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Second, do the Edman degradation.
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It is necessary to break large proteins down
into smaller pieces. The smaller pieces are then
sequences and the sequences are put
together. Methods of cleavage 1) Cyanogen
bromide 2) Enzymes Trypsin Chymotrypsin
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1) Cyanogen bromide cleavage.
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• Enzymatic cleavage.
• Trypsin cleaves on the C-terminal side of
  lysine or argining.

You determine the sequence of amino acids within
each peptide but not the sequence of amino acids
within the peptide. Use a second enzyme on the
original peptide.
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Chymotrypsin cleaves on the C-terminal side of
large hydrophobic amino acids.
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Put it together by comparing sequences.
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Other useful techniques. Dinitrofluorobenzene
Fred Sanger Nobel prize.
R1 O R2 O
NH2-CH - C NH-CH
C -aa2-aa3 etc.

F
NO2
NO2
R1 O R2 O
NH-CH - C NH-CH C
-aa2-aa3 etc.
HF
NO2
NO2
hydrolyze
-aa1 aa2 aa3
NO2
NO2
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Determine the C-terminal amino acid using
carboxypeptidase.
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Determining the sequence of amino acids in the
protein. B. Sequence the DNA - Sanger
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What determines the sequence of amino acids in a
protein? Can it be done by chance?
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20 amino acids can make thousands of possible
sequences.
5 amino acids.
Leu Asp Val His Gly
Leu-Asp-Val-His-Gly
His-Asp-Gly-Leu-Val is not the same peptide.
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How many possible peptides are there and what is
the probability of getting the one you want by
chance?
His-Asp-Gly-Leu-Val
His
Pick Aa 1 1/20
His-Asp
Pick Aa 2 1/20x 1/20 1/400
His-Asp-Gly Pick Aa3 1/29x1/20x1/301/8000
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What is responsible for the exact sequence?
The gene - DNA
Sequence of nucleotides determines the genetic
code. What happens if there is a mistake in
the DNA? mutation.
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• Summary
• The DNA provides the blueprint
• for the amino acid sequence (primary
• structure).

2.The amino acid sequence determines how the
protein folds in space (conformation)
(secondary, tertiary quaternary structure)
3. The conformation of the protein
determines its activity.