Cell Bio Final

• Amino group


  Accepts a proton
• Carboxyl group


  Donates a proton
• Peptide bonds


  1. Link amino acids with condensation reaction

  2. Charges on amino and carboxyl groups disappear
• 1. terminus


  First amino acid in polypeptide backbone

  
• 1. terminus


  Last amino acid in polypeptide backbone

  
• Side chain (R group)


  • Attached to the ⍺-carbon
  • Structure of side chain is what distinguishes one amino acid from another
• Types of amino acids


  • Polar (hydrophilic, form hydrogen bonds, interact with water)
  • Nonpolar (hydrophobic, cannot form hydrogen bonds, repel water)

  
• Noncovalent bonds in protein structure


  • Hydrogen bonds
  • Electrostatic interactions
  • Van der Waals attractions

  
• Noncovalent bonds between amino acids influence three dimensional shape of proteins

  
• Hydrophobic interactions play a critical role in determining shape of protein; nonpolar amino acids cluster in core

  
• Levels of protein structure


  • Primary (amino acid sequence)
  • Secondary (alpha-helices and beta sheets)
  • Tertiary (collection of secondary structures)
  • Quaternary (binding of different polypeptide chains)

  
• Disulfide bonds


  Covalent bonds between cysteines that stabilize protein structure or combine different polypeptide chains

  
• Disulfide bonds are often observed in proteins that are secreted by cell or are attached to outer surface of plasma membrane

  
• Ligand


  Any substance that is bound by a protein
• Binding site


  Cavity with amino acid side chains that bind to ligand using noncovalent bonds
• Binding sites often use electrostatic interactions and hydrogen bonds to selectively bind to one specific ligand
• Antibodies


  Proteins composed of four polypeptide chains; two identical light and two identical heavy chains
• Antibody variable regions


  VH and VL can bind to an antigen (substance that stimulates immune response)
• Producing antibodies in the lab


  1. Protein/antigen is injected into animal; antibodies that bind to antigen are secreted into blood by B cells

  2. Blood is collected and antibodies are purified
• Polyclonal antibodies


  Mixture of antibodies that bind to different places on same antigen (different antibodies made by different B cells)
• Producing monoclonal antibodies


  1. Step one: inject mouse with antigen to stimulate immune response

  2. Step two: isolate B cells from spleen from mouse

  3. Step three: fuse B cells with myeloma cells; hybrid cells produce antibodies but do not have cell senescence (immortal)

  4. Step four: identify which hybrid cells produce effective antibody
• Monoclonal antibodies


  Purified from hybrid cells; have low variability between batches (ideal for research and drug treatment)
• Protein denaturation


  Change in protein shape that reduces protein activity
• Protein denaturation is caused by heat, extreme pH, and chemicals
• Most proteins cannot spontaneously refold once they have been completely denatured
• Chaperones


  Cell use chaperones to refold proteins that have become denatured or are not properly folded

  
• Heat shock proteins (Hsp)


  Chaperone proteins that refold proteins that have been denatured (often due to an increase in temperature)
• Protein folding by Hsp70


  1. Hsp70 binds to segment of 4-5 nonpolar/hydrophobic amino acids

  2. Hydrolysis of ATP allows Hsp70 to bind to hydrophobic amino acids

  3. Hsp70 can bind to protein as soon as it is translated by ribosome

  4. Binding of Hsp70 to protein causes hydrophobic segment to extend

  5. Rebinding of ATP releases segment in extended form

  6. Repeated cycles of bind, extend, release helps protein fold properly

  7. Hsp70 is constantly extending polypeptide chain until enough protein is made for correct folding

  8. Hsp70 can also refold a misfolded protein already translated

  
• Protein folding by Hsp60

  1. Hsp60 is a multiprotein complex (Hsp60 x 14 + accessory proteins)

  2. Hsp60 Has 2 folding chambers

  3. Hsp60 folds proteins using ATP

  4. Hsp60 works in partnership with Hsp70 (delivers unfolded protein)

  
• Proteins that cannot fold properly need to be degraded to prevent protein aggregation

  
• Three steps for ubiquitylation


  1. Ubiquitin added to ubiquitin-activating enzyme (E1)

  2. Ubiquitin on E1 transferred to ubiquitin-conjugating enzyme (E2)

  3. E2 binds to ubiquitin ligase (E3); ubiquitin ligase adds ubiquitin to target protein and process is repeated to produce polyubiquitin chain
• Protein is denatured by cap, degraded in cylinder; ubiquitin and amino acids are recycled

  
• Each cell has about ~16,000 different proteins
• E1, E2, E3, proteins work together to target specific proteins
• Cells use combinations of E1, E2, and E3 to target proteins
• Cells activate a specific set of E1, E2, and E3 enzymes to target a specific protein (substrate)
• Cell fractionation


  1. Step one: collect desired cells

  2. Step two: lyse cells

  3. Step three: cell fractionation
• Cell lysate or cell homogenate


  Plasma membranes disrupted, sample has all cellular components (nuclei, organelles, membranes, etc.)
• SDS-PAGE


  Separates proteins in a mixture by size

  
• Beta-mercaptoethanol (BME)


  Reducing agent that eliminates disulfide bridges