Biotechnology Chapter Five Lecture- Proteins (part a)

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Biotechnology Chapter Five Lecture- Protein Structure and SDS-PAGE

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Biotechnology Chapter Five Lecture- Proteins (part a)

  1. 1. Introduction to Studying Proteins Chapter 5
  2. 2. Learning Outcomes ✤ Describe the structure of proteins, including the significance of amino acid R-groups and their impact on the three-dimensional structure of proteins. ✤ Explain the steps of transcription and translation in protein synthesis. ✤ Discuss the role of naturally occurring proteins and recombinant proteins in biotechnology. ✤ Differentiate proteins that function as part of structure, as antibodies, and as enzymes.
  3. 3. Learning Outcomes ✤ Describe the structure of antibodies and explain the relationship between antibodies and antigens. ✤ Discriminate among the classes of enzymes and discuss the effect of reaction conditions on enzyme activity. ✤ Summarize polyacrylamide gel electrophoresis and identify its usefulness for studying proteins.
  4. 4. 5.1 The Structure and Function of Proteins Virtually all biotechnology products have something to do with proteins. Protein function is determined by the three-dimensional structure(shape) of the protein.
  5. 5. 5.1 The Structure and Function of Proteins Virtually all biotechnology products have something to do with proteins. Protein function is determined by the three-dimensional structure(shape) of the protein.
  6. 6. Protein Molecule Structure Proteins are polymers composed of amino acids amino group carboxylic acid group
  7. 7. Twenty different kinds of amino acids are found in proteins
  8. 8. Protein Molecule Structure Most proteins contain tens of hundreds of amino acids chained together by peptide bonds. Molar mass and the specific amino acid sequence can be determined by mass spectrometry
  9. 9. Protein Molecule Structure Most proteins contain tens of hundreds of amino acids chained together by peptide bonds. Molar mass and the specific amino acid sequence can be determined by mass spectrometry
  10. 10. Protein Molecule Structure
  11. 11. Protein Molecule Structure Primary protein structure is the linear amino acid sequence including disulfide bridges between cystines. covalent bonds
  12. 12. Protein Molecule Structure Secondary protein structure is the folding and twisting that varies depending on the amino acid side chains. hydrogen bonds
  13. 13. Protein Molecule Structure Secondary conformations include the alpha helix and the beta pleated sheet
  14. 14. Protein Molecule Structure Secondary conformations include the alpha helix and the beta pleated sheet
  15. 15. Protein Molecule Structure Tertiary protein structure is the total 3-D shape. hydrogen bonds, ionic bonds, and hydrophobic interactions
  16. 16. Protein Molecule Structure Tertiary protein structure can be predicted from amino acid sequences or determined by x-ray crystallography.
  17. 17. Protein Molecule Structure Quaternary protein structure results from combining more than one polypeptide.
  18. 18. Function of Structural Proteins STRUCTURE = FUNCTION Viral recognition proteins Glycoprotein 120 on the surface of HIV, must exactly match its human cell membrane receptors to recognize, attach, and infect a T-helper cell.
  19. 19. Because proteins are often modified after transcription and they have complex 3-D structures, studying protein is much more challenging than studying DNA.
  20. 20. GFP protein
  21. 21. GFP protein is used extensively in biotech as a marker. When attached to a molecule of interest it glows!
  22. 22. GFP protein 239 aa 28,870 Daltons Dalton = 1 H atom Dalton = 1 amu KDa = 1000amu
  23. 23. BIO-RAD’s GFP has 3 mutations which allow for higher solubility in water.
  24. 24. Proteomics = study of proteins
  25. 25. Proteome = entire collection of an organism’s proteins
  26. 26. Separation and identification of proteins using SDS-PAGE Sodium DodecylSulfate = detergent used to denature protein and equalize amino acid charges PolyAcrylamide Gel Electrophoresis separates proteins by size and sometimes shape. http://www.jove.com/video/758/electrophoreticseparation-of-proteins
  27. 27. Separation and identification of proteins using SDS-PAGE Samples are mixed in Laemmli sample buffer containing Tris buffer- keeps pH 6.8 for electrophoresis Glycerol- weighs sample down for loading Bromophenol blue- colors sample *SDS *dithiothreitol (DTT)
  28. 28. Separation and identification of proteins using SDS-PAGE SDS Protein charges vary depending on the specific amino acid sequence. SDS equally coats all proteins in negative charge so that migration is only based on protein size.
  29. 29. Separation and identification of proteins using SDS-PAGE *DTTbreaks -S-S- bridges creating a linear polypeptide so migration is based only on size. can be left out to provide info on disulfide bond locations
  30. 30. Separation and identification of proteins using SDS-PAGE Heat is often used to ensure complete denaturing
  31. 31. Separation and identification of proteins using SDS-PAGE protein standards of known mass are included for comparison
  32. 32. Vocabulary • X-ray crystallography – a technique used to determine the three-dimensional structure of a protein • Polar – the chemical characteristic of containing both a positive and negative charge on opposite sides of a molecule • Primary structure – the order and type of amino acids found in a polypeptide chain • Secondary structure – the structure of a protein (alpha helix and beta sheets) that results from hydrogen bonding
  33. 33. Vocabulary • Tertiary structure – the structure of a protein that results from several interactions, the presence of charged or uncharged “R” groups, and hydrogen bonding • Quaternary structure – the structure of a protein resulting from the association of two or more polypeptide chains
  34. 34. 5.1 Review Questions 1. 2. How many different kinds of amino acids are found in proteins? What distinguishes one amino acid from another? What causes polypeptide chains to fold into functional proteins?
  35. 35. Questions and Comments?

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