MCB 102 Lec 6 Protein sequence

  1. What are the 4 levels of protein structure?
    • Primary
    • Secondary
    • Tertiary
    • Quaternary
  2. What is the primary structure of proteins?
    • Amino acid residues
    • Amino acids linked by peptide bonds to form peptides or proteins
  3. What is the secondary structure of proteins?
    • α-helix
    • β sheets
  4. What is the tertiary structure of proteins?
    Polypeptide chain
  5. What is the quaternary structure of proteins?
    Assembled subunits
  6. Amino acids are linked to other amino acids by what kind of bonds?
    Peptide bonds
  7. Describe peptide bonds
    • Very stable
    • Half-life is approximately 70 years at neutral pH
    • Peptide bond hydrolysis requires acid or base, plus heat
  8. What is the average molecular weight of an amino acid in a peptide or protein?
    Considered to be 110
  9. Proteins with different function vary greatly in what way?
    Vary in composition
  10. What makes a protein polymorphic?
    Difference in sequence when comparing across different organisms from the same species
  11. What are the proteins called that also include non-amino acid components?
    Conjugated proteins
  12. Who sequenced insulin?
    • Frederick Sanger
    • 1953
  13. Why was the sequencing of insulin significant?
    • It was thought to be impossible
    • Also the year Watson and Crick figured out the DNA structure
    • The relationship between proteins and DNA soon became clear
  14. Why is it important to know the sequence of the protein we are studying?
    Essential to further biochemical analysis
  15. The actual sequence of a protein is generally determined from what?
    DNA sequence
  16. What are the two methods of studying the primary structure of proteins/peptides?
    • Edman Degradation (Classical method)
    • Mass Spectrometry (Modern method)
  17. What is the classical method of studying the primary structure of proteins/peptides?
    Edman Degradation
  18. What is the modern method of studying the primary structure of proteins/peptides?
    Mass Spectometry
  19. What is Edman Degradation?
    Successive rounds of N-terminal modification, cleavage, and identification
  20. What is Edman Degradation used for?
    Can be used to identify protein without known sequence
  21. What is mass spectrometry?
    MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence
  22. What is mass spectrometry used for?
    Can be used to determine post-translational modifications
  23. What How did Sanger sequence insulin?
    • Amino terminal amino-acid residue is first labeled with a compound like 1-fluoro-2,4-dinitrobenzene (FDNB), that won't denature the protein
    • Acid is then used to hydrolyze the peptide bond (acid hydrolysis)
    • Partial acid hydrolysis will produce peptides of specific length, which can be separated
    • This process can be repeated
  24. What is Edman's Degradation?
    • Peptide bond nearest the amino terminus of the protein or polypeptide is cleaved in two steps
    • Two steps are carried out under very different reaction conditions
    • This allows one step to proceed to completion before the second is initiated
  25. What are the conditions for the two steps in Edman's Degradation?
    • Basic conditions in step 1
    • Acidic conditions in step 2
  26. What what point does Edman's degradation become inefficient?
    For peptides w/ >50 AAs
  27. How can disulfide bonds be broken?
    Through oxidation or reduction
  28. What can be used in the reduction process of breaking disulfide bonds?
    Dithiotheitol (DTT) or β-mercaptoethanol
  29. Why is the second reaction in the reduction process of breaking disulfide bonds necessary?
    To prevent the reformation of the disulfide bond
  30. What do proteases do?
    Catalyse the hydrolytic cleavage of peptide bonds
  31. What are the only kinds of peptide bonds that most proteases cut?
    Bonds that are adjacent to a particular amino acid residue
  32. Why do many proteases only cut a peptide bond that is adjacent to a particular amino acid residue?
    They fracgment the peptide in a reproducible and predictable manner
  33. Where does trypsin cleave?
    • Lys
    • Arg (C)
  34. Where does chymotrypsin cleave?
    Arg (C)
  35. Where does V8 protease cleave?
    • Asp
    • Glu (C)
  36. Where does pepsin cleave?
    • Leu
    • Phe
    • Trp
    • Try (N)
  37. Where does cyanogen bromide cleave?
    Met (C)
  38. Currently, what processes have yielded millions of protein sequences?
    Genetic sequencing and translation using DNA code
  39. What does mass spectrometry measure?
    Provides a highly accurate measure of the molecular weight of a protein
  40. What does mass spectrometry provide?
    • A highly accurate measure of the molecular weight of a protein
    • Specific sequences of short polypeptide segments
  41. Previously, why couldn't mass spectrometry be applied to macromolecules like proteins and nucleic acids?
    Because they would be destroyed when heated to the gas phase
  42. What were some new methods of mass spectrometry that could be applied to macromolecules?
    • MALDI MS
    • ESI MS
  43. What is MALDI MS?
    • Matrix-associated laser desorption
    • Proteins are placed in a light absorbing matrix
    • A short pulse of laser light, the proteins are ionized and desorbed from the matrix into the vacuum system
  44. What is ESI MS?
    Electrospray ionization mas spec
Card Set
MCB 102 Lec 6 Protein sequence
MCB 102 Lec 6 Protein sequence