CELLULAR BIOCHEMISTRY

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CELLULAR BIOCHEMISTRY PROTEINS AND
ENZYMES LECTURE 2 PROTEINS - PRIMARY
STRUCTURE LECTURER Mike Wallis - Room JMS 2C36
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FOUR LEVELS OF PROTEIN STRUCTURE

• PRIMARY
• SECONDARY
• TERTIARY
• QUATERNARY

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LECTURE 2 PROTEINS - PRIMARY STRUCTURE

• AMINO ACIDS
• THE PEPTIDE BOND
• PEPTIDES, POLYPEPTIDES AND PROTEINS
• SEQUENCE DETERMINATION
• APPLICATIONS OF SEQUENCES

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AMINO ACIDS

• The building blocks of proteins
• Released from proteins by hydrolysis
• (e.g. with 6M HCl, 110ºC, 24h)
• Separated by chromatography or
  electrophoresis
• Detected by reaction with ninhydrin (purple
  colour)

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AMINO ACID STRUCTURE
a zwitterion
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Stereoisomerism (optical isomerism) of amino acids
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Separation of amino acids by paper chromatography
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Separation of amino acids by ion exchange
chromatography
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NON-POLAR AMINO ACIDS
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POLAR AMINO ACIDS
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THE PEPTIDE BOND
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THE HISTORY OF PROTEIN SEQUENCE DETERMINATION

• 1945 - 1955 Insulin sequence determined by
  Sanger and colleagues
• 1963 the first enzyme sequence - Ribonuclease -
  Moore and Stein
• 1963 - present hundreds of protein sequences
  determined
• 1977 - first RNA/DNA coding sequences described
  - protein sequences deduced from these
• 1990s Complete genome sequences of many
  prokaryotes, yeast, C.elegans and Drosophila
  reported. Protein sequences of corresponding
  genes deducible?
• 2000 Initial human genome sequence reported.
  Sequences of all human proteins deducible?

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STEPS REQUIRED IN DETERMINATION OF AMINO ACID
SEQUENCE
1. Preparation of pure protein 2. Amino acid
analysis 3. Determination of N- and C-terminal
amino acids 4. Separation of polypeptide chains
if more than one 5. Determination of amino acid
sequence using Edman Degradation 6. For each
chain, break into smaller fragments, fractionate
and characterise 7. Break chain using second
cleavage method, separate and characterise
peptides 8. Assemble peptides into unique
structure 9. Assign disulphide bridges (if
present)
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Amino acid sequence of bovine growth hormone
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Amino acid sequence of ribonuclease
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USES OF AMINO ACID SEQUENCES
1. Demonstration that protein sequences are
unique 2. Facilitate studies on
structure-function relationships, protein
engineering and 3D stucture determination 3.
Provide a basis for genetic studies - genetic
code, mutations etc. 4. Provide a basis for
studies on molecular evolution. protein
families. Molecular phylogeny. 5. Allow
chemical synthesis of proteins and genes 6.
Potentially allow prediction of 3D structure 7.
Allows analysis of post-translational
modification
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SICKLE CELL HAEMOGLOBIN
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POST-TRANSLATIONAL MODIFICATION

• Several amino acid side chains in proteins may be
  modified after protein synthesis has occurred.
  For example
• Ser, Thr and Tyr are sometimes phosphorylated
• Asn and Ser are sometimes glycosylated
• CySH is sometimes modified by attachment of a
  lipid anchor