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  1. X-Ray Crystallography
    • A beam of parallel X-rays is aimedat a crystal of protein molecules
    • Electrons in crystal diffract the x-rays, which are then recorded on film or by an electronic detector
    • Mathmatical analysis of the pattern produces an image of the electron clouds surrounding atoms and the positions of each of the atoms in 3D space
  2. X-Ray Chromotography Problems
    • Originally used to study the simple repeating units of fibrous proteins and the structures of small biological molecules
    • Difficult to calculate atomic positions from the positions and intensities of diffracted x-ray beams
    • Difficult to prepare crystals of a quality suitable for x-ray diffraction 
  3. X-Ray Crystallography: Protein Crystal
    • Contains a large number of water molecules
    • Possible to diffuse small ligands such as substrate or inhibiotr molecules into the crystal
    • The proteins within the crystal retain their ability to bind these ligands and exhibit catalytic activity
    • -Demonstrates that the proteins crystallize in their in vivo native conformations 
  4. Space-Filling Models
    • Depict each atom as a solid sphere
    • Reveal the dense, closely packed nature of folded polypeptide chains
    • Used to illustrate the overall shape of a protein and the surface exposed to aqueous solvent
    • Interior of folded proteins is nearly impenetrable, even by small molecules like water 
  5. Simplified Cartoon
    • Emphasizes the backbone of the polypeptide chain
    • The amino acid side chains have been eliminated, making it easier to see how the polypeptide folds into a 3D structure
    • Advantage of allowing us to see into the interior of the protein
    • Reveal elements of secondary structure such as alpha helices and beta strands
    • Comparing the structures of different proteins is possible to recognize common folds and patterns that can't be seen in space-filling models
  6. The structures of the amino acid side chains
    Various covalent bonds and weak interactions between atoms 
    • Best type of model to show details focus on:
    • Important to understand how a substrate binds in the active site of an enzyme 
  7. NMR Spectroscopy
    • Permits the study of proteins in solution
    • A sample of protein is placed in a magnetic field
    • Certain atomic nuclei absorb electromagnetic radiation as the applied magnetic field is varied
    • Absorbance is influenced by neighboring atoms, so interactions between atoms that are close together can be recorded
    • Combining these results with the amino acid sequence and known structural constraints, it is possible to calculate a number of structures that satisfy the observed interactions
    • Only works with small proteins
  8. Alpha Helix
    • Repeat of 0.5-0.55nm is the pitch (axial distance per turn)
    • 0.15nm is the rise (distance each residue advances along it's axis)
    • Present in hemoglobin
    • Almost always right-handed
    • Ideal one requires 3.6 amino acid residues
    • Hydrogen bonds between amino acid residues are stable in the hydrophobic interior, because water is not in it to compete for hydrogens
  9. Residues
  10. Alpha Helix
    • Peptide group is polar
    • Dipole with +N-terminus and -C-terminus
    • Stability is affected by the identity of the side chains 
    • Alanine very common
    • Tyrosine and asparagine less common
    • Glycine destabilizes them, many begin or end them
    • Proline is least common because it lacks an H+ on its amide nitrogen
    • Have a hydrophilic at one end of helix and hydrophobic at the other end 
  11. Beta Strands
    • Portions of the polypeptide chain that are almost fully extended
    • Multiple for beta sheets 
  12. Beta Sheets
    • Stabilized by hydrogen bonds between carbonyl oxygens and amide hydrogens on adjacent beta strands
    • When antiparallel, hydrogen bonds are perpendicular to the extended polypeptide chains
    • Parallel is less stable than antiparallel
    • Forms fully in quaternary structure 
  13. Loops
    • Contain hydrophilic residues
    • Found on the surface of proteins where they are exposed to solvent and form hydrogen bonds with water 
  14. Turns
    • Loops containing only a few residues if they cause an abrupt change in the direction of a polypeptide chain
    • Up to 5 
  15. Reverse Turns
    • Connect different antiparallel beta strands
    • 2 types: type I and type II

  16. Type I & II Reverse Turns
    • Contain 4 amino acid residues
    • Stabilized by hydrogen bonding between the carbonyl oxygen of the 1st residue and the amide H+ of the 4th residue
    • Produce an abrupt change in the direction of the polypeptide chain
    • Proline is often the second residue 
  17. Type II Reverse Turn
    • Third residue is glycine 
    • Abrupt change of backbone  
  18. Tertiary Structure of Proteins
    • Results from the folding of a polypeptide into a closely packed 3-D structure
    • Amino acid residues that are far apart in the primary structure are brought together, permitting interactions among their side chains
    • Stabilized by noncovalent interactions between the side chains of amino acid residues
    • Disulfide bridges 
  19. Motifs
    • Supersecondary structures
    • Combinations of alpha helices, beta strands, and loops that appear in a number of different proteins
    • Associated with a particular function
    • Most common is helix-loop-helix
    • Coiled coil
    • Helix bundle
    • Beta-alpha-beta unit
    • Hairpin
    • Beta meander
    • Greek key
    • Beta sandwich 
  20. Helix-Loop-Helix Motif
    • Occurs in a number of calcium-binding proteins
    • -Glutamate and aspartate residues in the loop of these proteins form part of the site
    • Form a reverse turn
    • Residues of alpha helices bind DNA
  21. Coiled-Coil Motif
    • Consists of 2 amphipathic alpha helices that interact through their hydrophobic edges
    • Leucine zipper
    • Individual alpha helices are parallel
  22. Helix Bundle Motif
    • Several alpha helices can associate to form one
    • Individual alpha helices have opposite orientations
  23. Beta-Alpha-Beta Motif
    • Has 2 parallel beta strands linked to an intervening alpha helix by 2 loops
    • Helix connects the C-terminal end of one beta strand to the N-terminal end of the next
    • Runs parallel to the 2 strands 
  24. Hairpin Motif
    Has 2 adjacent antiparallel beta strands connected by a beta turn
  25. Beta-Meander Motif
    • An antiparallel beta sheet composed of sequential beta strands connected by loops or turns
    • The order of strands in the beta sheet is the same as their order in the sequence of the polypeptide chain
    • May contain one or more hairpins, but normally are connected by larger loops 
  26. Greek Key Motif
    A beta sheet motif linking 4 antiparallel beta strands 3 and 4 form the outer edges of the sheet and strands 1 and 2 are in the middle of the sheet
  27. Beta-Sandwich Motif
    Formed when beta strands or sheets stack on top of one another 
  28. Domains
    • Discrete, independently folded, compact units
    • May consists of combinations of motifs
    • Consists of various elements of secondary structure
    • Connected by loops, but also bound to each other through weak interactions formed by amino acid side chains on the surface 
  29. Fold
    Combination of secondary structures that form the core of a domain
  30. Quaterynary Structure of Proteins
    • The organization and arrangement of subunits in a protein with multiple subunits
    • -Each subunit is a separate polypeptide chain
    • Hydrophobic interactions are the principal forces involved
    • Electrostatic forces may contribute to the proper allignment of the subunits
  31. Quaternary Structure: Oligomers
    • Normally more stable than their dissociated subunits, suggesting it prolongs the life of a protein in vivo 
    • The active sites of some of these enzymes are formed by residues from adjacent polypeptide chains
    • 3D structures of these change when the proteins bind ligands
    • -Structures of the contacts between subunits may be altered
    • Different proteins can share the same subunits 
  32. Denaturation
    • Environmental changes or chemical treatments that disrupt the native conformation of a protein, with concomitant loss of biological activity
    • The amount of energy needed is usually small
    • Retain considerable internal structure
  33. Myoglobin and Hemoglobin
    • Aerobes
    • Multitissue organisms
    • Oxygen nonpolar, low water solubility, so have oxygen transport (hemoglobin)/storage (myoglobin) system
    • Oxygen not bound by proteins, need a “helper” ligand, and a prosthetic group:
    • -Apoprotein binds prosthetic group to make holoprotein
    • Signal (energy): RECEPTORS NECESSARY
    • Prosthetic group is heme
    • -Water soluble, and can bind oxygen by itself
  34. Proteins and Iron
    • The iron in the heme must be ferrous iron, but oxygen will oxidize the ferrous iron to ferric, which will not bind oxygen
    • Solution: oxidation requires 2 hemes to form an oxygen sandwich and the proteins
    • The iron requires 6 ligands
    • - 4 provided by the tetrapyrrole ring
    • - 2 provided by the protein, 2 histidyl side chains
    • In myoglobin, one is near and the other is farther away
    • The distal 
  35. Hemoglobin
    • Has 4 globins and hemes. Its globins are nearly identical in folding to myoglobin
    • A heteroligomer
    • - 2 alpha and 2 beta subunits
    • Hemoglobin A, HbA, is the most common form
    • Fetal hemoglobin, HbF, is replaced by HbA after birth
    • Sickle cell hemoglobin, HbS, has one E in the beta chain replaced by a V, producing a “sticky” patch on the surface of the protein
    • -This causes the HbS to polymerize
  36. There is a pH-dependence, call the Bohr effect
    There is dependence on a metabolite, 2,3-biphosphoglycerate
    Dependence on a waste product, CO2
    3 Reasons there's a curve in the hemoglobin shape 
  37. Purpose of Interaction in Hemoglobin
    Weaken binding of oxygen so hemoglobin unloads oxygen more completely in tissues so myoglobin can bind it
  38. Interaction in Hemoglobin
    • The ferrous iron is actually too big for the hole in the tetrapyrole ring- until oxygen binds
    • Then it moves into the hole, moving the proximal H
    • Polypeptide chains are at Van Der Waal’s distances, so movement of the proximal H is transmitted THROUGHOUT the oligomer
    • -The 2 alpha beta pairs rotate 15 degrees
    • -This closes a central cavity lined with positively charged residues
  39. Bisphosphoglycerate has 3-5 negative charges
    Tissues are producing acid, so the blood pH in tissues is lower, 7.2
    CO2, also produced in the tissues, also stabilizes the deoxy form by reacting nonenzymatically with the amino termini to form carbamates
    Subunit movement on oxygen binding has 3 effects in tissues
  40. Bisphosphoglycerate
    • 3-5 negative charge
    • It CANNOT bind in the central cavity to oxyhemoglobin (lungs), but in the tissues, the vaity opens when oxygen comes off, so the bisphosphoglycerate stabilizes the deoxy form
    • Interactions between sites biding different ligands is called alloterism

  41. Tissues Produce Acid
    • The blood pH in tissues is lower, 7.2
    • This “Bohr effect” weakens oxygen binding in tissues
    • Chemical basis: the C-terminal residue of the beta chains is 146H. It’s carboxyl forms a salt bond with the amino of 40K of the alpha chains. This keeps the imidazole of 146H near the side chain carboxyl of D94, beta chain
    • When the imidazole is protonated, another salt bond forms
  42. When the imidazole is protonated, another salt bond forms: 3 effects
    • New salt bond raises the pK of H146, as it stabilized the protonated form
    • New salt bond stabilizes the deoxyhemoglobin form
    • The hemoglobin is now carrying acid from the tissues to the lungs
  43. CO2
    • Produced in tissues
    • Stabilizes the deoxy form by reacting nonenzymatically with the amino termini to form carbamates
    • These can form salt bonds with 141R (residue), alpha chains
    • 3 effects:
    • Lowers the pH of the amino termini
    • Aids oxygen release in tissues, because stabilizes the deoxy form
    • -The deoxyhemoglobin is now carrying CO2 from the tissues to the lungs
    • In the lungs, the acid and bicarbonate combine to produce CO2, which is expelled
    • -The organization to this one level is down to a protein

  44. Enzymes
    • Great catalytic power
    • Specificity
    • -Type of reaction
    • -Compounds operated on: “substrates”
    • Organized into pathways
    • Pathway rates under control
    • Control energy transformation
    • -Photosynthesis
    • -Storage for ATP
  45. Oxidoreductases
    -Lactic dehyrogenase (Ldh)
    Enzymes divided in 6 classes based on type of reaction catalyzed 
  46. Chemical Kinetics of Enzymes
    • Mainly determine order of reaction- clue about mechanism; zero, first, second
    • Muscle contraction
    • Enzyme converts chemical energy to mechanical energy in lightning bugs
    • Any chemical reaction may be written
  47. Enzyme Kinetics
    • A branch of chemical kinetics
    • Sucrase, a hydrolase
    • Glucose is a reducing sugar (has an aldehyde), so it can be measured
  48. Enzyme Kinetics Convention
    • E= enzyme
    • S= substrate
    • P= product
    • So, E+S gives E+P
    • E is unaltered
    • In cells, E is near S
    • In the lab, S is in great excess
  49. Inhibition Kinetics
    • Used in drug design
    • To make better inhibitors for HIV protease
    • Also to study regulatory enzymes, as regulators almost always affect the kinetics of the enzymes they regulate
  50. Diffusion-Controlled Reactions
    • Rate of substrate binding (K1) limiting, not Kcat
    • Do not show Michaelis-Menten kinetics
    • Very high Kcat/Km values
    • Examples
    • -Superoxide dismutase: Kcat/Km= 2^109 M-1sec-1
    • -Triose phosphate isomerase: Kcat/Km= 4^108 M-1sec-1
  51. Chemical Modes of Catalysis
    • Acid-base catalysis
    • Covalent catalysis
    • Chemical model studies show each mode gives rate enhancements of 10 to 100
  52. Binding Modes of Catalysis
    • Experimentally demonstrable
    • The “proximity effect”- binding of substrates close to other substrates or reactive side chains
    • Increases effective concentrations of reactants
    • Converts second-order reactions to first-order
  53. Model Compounds of Catalysis
    • Up to 10^8-fold rate enhancement
    • Major effect, yet not sufficient
    • Transition state stabilization
    • - Preferential binding of analogues though to resemble “transition state” 
Card Set:
2012-06-24 18:45:34

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