m21 - Protein Sequence Determination It is useful to know...

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Protein Sequence Determination: Edman Degradation It is useful to know the specific amino acid sequence of a protein Edman degradation: method developed in the 1950s to determine protein sequence With genome sequencing, this method is not as widely used. Protein sequence can be deduced indirectly from the DNA sequence based on the genetic code
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NH NH 2 - K - S - L - V - E- E- COOH - K - S - L - V - E- E- COOH cycle 1 cycle 1 PITC coupling PITC coupling -NH- K - S - L - V - E- E- COOH Acid cleavage Acid cleavage -NH-K -NH-K NH2 -S - L - V - E - E - COOH NH2 -S - L - V - E - E - COOH + K Amino acid Amino acid analysis Repeat for cycle 2, cycle 3, etc. Repeat for cycle 2, cycle 3, etc. Sequence the order of the peptide at the N-terminus ~30 aa can be determined Phenylisothiocyanate (PITC) Edman degradation
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Approximately 30 or so amino acids can be clearly determined. To sequence an entire protein, small sections have to be determined and then pieced together Start with a purified protein You can use proteases that cleave bonds between specific amino acid pairs Generates a small protein puzzle where the pieces can be put together to determine the order of the fragments Edman Degradation
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•These can then be subjected to Edman degradation. • The entire protein sequence can be pieced together only after sequencing of two sets of protease fragments. N C N N N N N C C C C C polypeptide smaller peptides protease Edman Degradation
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Basic Molecular Genetic Mechanisms Chapter 4, pp 101-108
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Overview of the four basic molecular genetic processes
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History Lesson: The Discovery of DNA as the Genetic Material
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Mmmm, Peas! Segregation of homologous chromosomes and independent assortment Notable Events in the Quest for DNA
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Discovery of DNA Nucleic acids A, T, G, C Proteins A, V, I, L, M, F, Y, W, K, R, H, D, E, S, T, N, Q, C, G, P 4 different building blocks 20 different building blocks •1860s: Gregor Mendel presents studies on heredity in peas •1869: Friedrich Miescher isolated large substance in nucleus of white blood cells in pus from surgical bandages »Slightly acidic, sugary, rich in phosphorus »Called it “nuclein” •1920s: Genetic materials are in chromosomes, which are in the nucleus »Chromosomes contain a variety of proteins in addition to DNA »Prevailing view among biologist during this time was that proteins were the chief carriers of heredity.
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1929: P. A. Levene determined the chemical composition and structure of the building blocks of DNA But the scientific world strongly believed that DNA is not as complex as protein to be the genetic material It wasn’t until a series of experiments performed by Frederick Griffith (1928), Avery, MacCleod, McCarty, Chargaff, and Hershey and Chase (1952) that convinced the scientific community that DNA, and not proteins, is the heritable, genetic material.
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  • Spring '06
  • staff
  • DNA, Frederick Griffith, genetic material, Circular DNA molecule

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