Exam 1

• Amino acids


  In water (pH 7), free amino acids exist in ionized form; amino group accepts a proton and carboxyl group donates a proton
• Amino acids


  • All amino acids have an amino group and carboxyl group connected by ⍺-carbon

  
• Peptide bonds


  Link amino acids with condensation reaction; charges on amino and carboxyl groups disappear

  
• N-terminus


  First amino acid in polypeptide backbone
• C-terminus

  Last amino acid in polypeptide backbone
• Amino acids


  • Each amino acid has a side chain (aka R group) attached to the ⍺-carbon

  
• Side chains


  Structure of side chain is what distinguishes one amino acid from another
• Side chain properties


  • Polar amino acids: hydrophilic amino acids that form hydrogen bonds and prefer to interact with water
  • Nonpolar amino acids: hydrophobic amino acids that cannot form hydrogen bonds and 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

  
• Protein domains


  Segment of amino acids that can perform specific function
• Levels of protein structure

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

  
• 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

  
• Noncovalent bonds allow different polypeptide chains to combine to make one large protein (quaternary structure)

  
• Protein function


  Protein shape dictates protein function
• 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
  • Have variable regions (VH and VL) that 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


  Purified mixture of antibodies that bind to different places on same antigen (different antibodies made by different B cells)

  
• Monoclonal antibodies


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

  
• 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

  
• 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)
  • Hsp70 and Hsp60 are two different heat shock proteins that are made by the cell in response to stress
• 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

  
• 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
• Cells use combinations of E1, E2, and E3 to target specific proteins (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