Biochem Ch 4, 5, & 6

  1. What two compounds are on amino acids and what are their pKas?
    • Amine (pKa ~9.5)
    • Carboxylic Acid (pKa ~2)
  2. Chiral molecules
    • A molecule that cannot be superimposed on its mirror image
    • Are optically active and rotate plane polarized light
    • Amino acids (except glycine) are chiral bc bond to the central carbon is different
  3. What stereoisomers are all proteogenic amino acids?
    L-stereoisomers
  4. L/D nomenclature
    • Based on the structure of L-glyceraldehyde
    • L: (-) Left
    • D: (+) Right
  5. Fischer Projections
    • Presents three-dimensional chemical structures in two-dimensions
    • Image Upload 1
  6. Greek Lettering of Carbons
    Image Upload 2
  7. What charges are the amine and carboxyl groups at physiological pH (7.4)?
    • Positively charged amine
    • Negatively charged carboxyl group
  8. What is the net charge of an amino acid at pH 7.4?
    Zero net charge- Zwitterion
  9. Zwitterionic form
    Image Upload 3
  10. Titrating Alanine
    Image Upload 4
  11. Small nonpolar amino acids
    • Glycine (G)
    • Alanine (A)
  12. Large nonpolar (hydrophobic)
    • Valine (V)
    • Leucine (L)
    • Isoleucine (I)
    • Methionine (M)
  13. Methionine vs other hydrophobic a.a.
    • Met's side chain is very flexible and can adjust to geometrical constrains of other residues
    • Often found in amphiphilic helices involved in protein-protein interactions
    • Easily (and reversibly) oxidized, so can be used to "protect"
  14. Aromatic AA
    • Phenylalanine (F)
    • Tyrosine (Y)
    • Tryptophan (W)
  15. Phenylketonuria (PKU)
    • Genetic Disorder characterized by a mutation in the gene for the hepatic enzyme phenylalanine hydroxylase (PAH)
    • Can lead to intellectual disability
    • Treatment: strict Phe-restricted diet supplemented by other amino acids
  16. Tryptophan (W)
    • Precursor of serotonin "happiness hormone"
    • Serotonin is increased by several antidepressant drugs and sunlight
    • Some psychedelics are agonists of this molecule
  17. Aromatic AAs and UV
    • Tyr and Trp absorbs UV light at 280 nm and most proteins contain either or both
    • Therefore, the protein can be determined based on detection of these
    • Both residues are also fluorescent. This can be used to probe conformational changes of unmodified proteins
  18. Beer-Lambert Law (Beer's Law)
    Image Upload 5
  19. Uncharged and polar
    • Cysteine (C)
    • Serine (S)
    • Threonine (T)
  20. Cysteine Oxidation
    • Curly vs. Straight Hair
    • Keratin contains cysteines and the cross linking provide hair stability and define straight or curly hairs
    • Image Upload 6
  21. Uncharged and polar AA
    • Asparagine (N)
    • Glutamine (Q)
  22. Charged side chains: Carboxylic Acids
    • Asparagine (N)
    • Aspartate (D)
    • Glutamate (E)
  23. Glutamate (E)
    • Important neurotransmitter (memory)
    • MSG is commonly used as taste enhancer
  24. Isoelectric Point (PI)
    • pH at which a particular molecule carries no net electrical charge
    • Determined as an average value
    • Image Upload 7
  25. Charged Side Chains: Basic
    • Lysine (K)
    • Arginine (R)
  26. What % of the histidine side chain is ionized at pH 7?
    • Image Upload 8
    • Image Upload 9
  27. How does environment affect actual pKa values?
    • Neighboring charges
    • Nonpolar environments
    • Hydrogen bonding
    • In enzyme active sites: pKa is often drastically different
  28. Beta branching
    • Valine
    • Isoleucine
    • Threonine
  29. 21st amino acid
    • Selenocysteine has a lower pka and reduction potential than cysteine which makes it suitable in proteins that are involved in antioxidant activity.
    • More active than S, present in several metabolic enzymes, mainly reductases and dehydrogenases
  30. How is selenocysteine encoded in genome if all codons are taken?
    Reprogramming the UGA "stop" codon- translational recoding
  31. 22nd amino acid
    • Pyrrolysine is encoded in Archaea by another "stop" codon-UGA
    • Helped the
  32. Peptide (amide) bond
    • Formed by the condensation of two amino acids accompanied by the release of a water molecule 
    • Bond specific to amino acids between the carboxylic acid and amine groups
  33. Peptide drawing rules
    • Always written N to C
    • If one-letter codes, all capital letters
    • If three-letter codes, first letter capitalized
    • Termini are sometimes specified
  34. Planarity of peptide bonds
    • Partial double bond character maintains planarity of peptide (amide) bonds
    • Protein backbone rotations are similar to sheets of paper
    • Planar because of the orbitals that form
    • In linear form side chains are on alternating sides but not extensively found in folded proteins
    • Image Upload 10
  35. Restraints on local peptide structure
    • Free rotation is limited to the NH-Calpha (Phi) and Calpha-CO (Psi)
    • Angles around these bonds are known as "dihedral" or "torsion" angles
    • Image Upload 11
  36. Cis amides
    • If both Calpha atoms on the same side of the peptide bond- the bonds are Cis
    • Image Upload 12
    • Too many interactions in the Cis position so not commonly found in nature
  37. Trans amides
    • If both the Calpha atoms are on the opposite sides of the peptide bond
    • Image Upload 13
  38. Which aa has similar energy for cis and trans conformations?
    • X-Pro
    • Due to the ring structure it is ~30% cis
    • Image Upload 14
  39. Extended conformation of psi and phi bonds
    • Side chains extend on opposite sides of the backbone
    • Full extended conformation is rare in protein structures
    • Easiest semi-realistic geometry on the plane of the paper
  40. Ramachandran Plot
    • Phi, Psi dihedral coordinates
    • Amino acid side chain determines allowed conformations
    • The allowed or favored (green) regions also correspond to the main types of secondary structure
    • Image Upload 15

    Glycine has more surface covered on the graph because there are no side chains
  41. In vivo peptide bond formation in ribosomal protein synthesis
    • Translation proceeds N to C
    • Carried out by ribosomes
  42. Non-ribosomal protein/peptide synthesis (i.e antibiotics)
    • Forms complex structures incorporating standard and modified amino acids (including d-amino acids)
    • Utilizes modular enzyme architecture
  43. Primary protein structure
    Linear sequence of amino acids linked by peptide bonds
  44. Secondary structure
    • alpha helices, beta sheets, loops & turns
    • Defined by local interactions of primary sequence
  45. Tertiary Structure
    Protein domains- defined by long range interactions between secondary structural units
  46. Quaternary structure
    Multi-subunit protein complexes- associations between two or more proteins
  47. Central Dogma
    • Protein primary sequence is determined by DNA and encodes all required information for protein to fold and function properly 
    • Image Upload 16
  48. AA sequence primary structure
    • Sequence of amino acids from N- to C- terminus 
    • May include disulfide bonds
  49. Oligopeptide
    Few amino acids linked
  50. Polypeptide
    Many amino acids linked
  51. Protein
    Large polypeptide, typically with MW>10,000 Da (g/mole) cutoff
  52. Protein secondary structure
    • Common repeating patterns of relative orientation of amino acid residues in a peptide in 3D space stabilized by hydrogen bonds
    • The stabilizing hydrogen bonds connect backbone elements of the polypeptide chain
  53. Common secondary structures
    • Alpha helix
    • Beta sheet
  54. Rules for secondary structures
    Stable secondary structural elements

    • Predicted and described structures thaat contained planar peptide bonds and were: 
    • 1) Sterically allowed and favorable
    • 2) Maximized the hydrogen bonding capacity of backbone amines and carbonyl groups
  55. Right-Handed alpha-helix
    • 3.6 residues per turn
    • 5.4 Pitch
    • i to i+4 hydrogen bonding pattern: C=O of residue and i and amide N-H of residue i+4
    • Phi= -57degrees; Psi= -47degrees
    • Hydrogen bond distance= 2.8
    • Image Upload 17
  56. What is the ideal alpha helix periodicity
    • 3.6 residues per turn, encloses 13 atoms in a ring by formation
    • 3.613-helix is more tightly and has 13 residues
  57. 310-helix
    • 3 is the residue with 10 turns
    • Is more tightly wound helix
    • Stabilized by i, i+3 bondsImage Upload 18
  58. Side chain orientation of right-handed alpha helix
    • Side chains point "out" and "back"
    • Very few steric clashes
    • Maximized hydrogen bonding of backbone amides
  59. Helical wheel layout
    • Electrostatic interaction across a turn of the helix
    • Side chains a and d (i and i+3) or a and e (i and i+4) are close in space
    • These interactions can stabilize or destabilize the helix
    • Image Upload 19
  60. Coiled-Coil helices
    • Are common
    • Occur when an amphipathic helix is primarily hydrophilic, but has hydrophobic residues in the i and i+3 positions which allow two helices to interact with each other
    • Can be further stabilized by interstrand salt bridges
    • Can be parallel or antiparallel; homo- or hetero-dimers
    • Image Upload 20
  61. Beta sheet bonding
    Antiparallel bonds are straight and stronger than parallelImage Upload 21
  62. Why are beta sheets favored?
    • By large hydrophobic residues 
    • They keep large and branched side chains far apart, minimizing steric clash
  63. Between alpha helix and beta sheet, which is more likely to form?
    Alpha helix are more likely to form since they are closer together and form faster
  64. Extended beta sheets
    In 3D these are twisted and arrows indicate N to C directionality
  65. Jellyfish and GFP
    • Green florescence protein which allow us to see other proteins in the cell
    • Tracking individual neurites (axons and dendrites) labeled by a combination of flurescent proteins
  66. Random Coil
    High conformational flexibility and no detectable structure
  67. Coil structure
    Any structured region that does not fit standard secondary definitions
  68. Turns and loops
    • Short secondary structural units that connect more standard units
    • Short beta hairpin turns
  69. Omega Loops
    Longer connecting segments (any number and sequence of amino acids)
  70. Beta hairpin turns
    • Generally connect antiparallel beta strands 
    • Can occur in isolation
    • Typically 4-5 amino acids stabilized by a hydrogen bond between residues i and i+3
    • Glycines and prolines are often found in beta turns
  71. Type I and II beta turns
    Stabilized by a hydrogen bond between residues i and i+3
  72. Gamma turns are stabilized by what?
    Hydrogen bonds between residues i and i+2
  73. Image Upload 22
    Image Upload 23
  74. Domains
    • Separate structural clusters within one protein chain 
    • One protein will have multiple domains
    • Some are very clearly separated and others are not as distinct
  75. Image Upload 24
    Quaternary structure
    Image Upload 25
  76. Subunit
    Separate protein chain
  77. Multisubunit complex
    Has multiple chains
  78. Massive quaternary assemblies
    Viruses: polio and tobacco mosaic virus
  79. Eukaryote massive quaternary assembly
    Vault a large cellular particle (organelle)- multiple copies of three proteins and RNA
  80. Polyribosomes
    When you have multiple synthesis site on mRNA
  81. Driving forces between protein forces
    Enthalpy and entropy
  82. Favorable entropic forces
    Reduced water cages (hydrophobic effect)
  83. Favorable enthalpic contributions
    • The chemical bonds between: 
    • H bonds betwene polar groups
    • Van-der-waals bond between hydrophobic sidechains (plentiful, but weak bonds)
    • Salt bridges (strong but few)
  84. Unfavorable entropic cost
    • Reduced configurational entropy
    • Just sheer folding is not favorable but the hydrophobic effect is greater than this so folding still occurs
  85. Unfavorable enthalpic contributions
    Desolvation cost: to form hydrogen bonds or salt bridges between polar groups within a protein in a folded state, the same groups should break bonds with water molecules in the unfolded state
  86. Folded (native) state
    • Buried hydrophobic groups
    • Shielded from water
  87. Unfolded (denatured) state
    • Unfavorable water cages
    • Entropic cost
  88. Amphipathic proteins
    • Protein backbones are polar
    • Polarity of side chains vary from very hydrophilic to very hydrophobic
    • Proteins fold to minimize contacts between hydrophobic residues and water (maximize intramolecular hydrophobic interactions)
  89. Hydropathy Plots
    Hydrophobicity of sequential regions can predict locationImage Upload 26
  90. Van der Waals forces
    • Are specific interactions between hydrophobic groups that stabilize a well-folded protein interior
    • Weak enthalpic effect
  91. The key to defining protein structure
    • Hydrogen bonds and are necessary to remain stable
    • It is also one of the major forces in protein stability
  92. How is that possible if the number of broken bonds with H2O (Hunfold) is equal or lower that the number of intra molecular bonds within the folded protein (Hfold)?
    Hydrogen bonds are often stronger between protein groups that between protein and water
  93. Salt bridges
    • Electrostatic interactions in proteins
    • Specify combined electrostatic and hydrogen bondings
    • Recovers approx. the same energy as the cost of taking charges out of water
  94. Interior vs exterior salt bridges
    • Interior: contribute to stability and conformational specificity
    • Exterior: charged residues contribute specificity for interaction with other proteins and ligands
  95. Covalent disulfide bonds
    • May stabilize protein structures when not in a reducing environment (cytoplasm is reducing)
    • Stability of small proteins may depend on these
    • Larger proteins may retain fold in absence of disulfides
  96. Protein Denaturation
    • Unfolded proteins, or proteins in which the secondary and tertiary structure has been disrupted to create random coil, are termed "denatured"
    • Can be caused by chemical or thermal means
  97. Chaotropes
    • Small molecules commonly used to denature proteins
    • Used in high concentrations
    • Bind water and reduce hydrophobic effect (can form many H bonds with water and decreases water cage)
    • Typically reversible 
    • Image Upload 27
  98. Reversible two-state protein folding
    Cooperative unfolding; when part of the structure is disrupted, it destabilizes the remaining structure
  99. Levinthal's Paradox
    • Do proteins fold through a random, sequential search pattern?
    • It takes approx 10-13 secs to rotate around a bond i.e to sample a possible protein backbone conformation
    • To sample all of the possible conformations of all the residues in a fairly small, 100-amino acid protein, it would take: 
    • Time to fold randomly 10n * 10-13s= 1087s
    • which is over 5 billion years
    • Proteins fold in milliseconds to seconds, sometimes minutes
    • Proteins do not fold by sequential random search
  100. Hierarchial model of protein folding
    • Structural
    • Local structures
    • Stabilization of secondary structure
  101. Structural protein folding
    • Rapid formation of local interactions (i.e. secondary structure), which then determine the further folding
    • These local interactions serve as nucleation points in the folding process
  102. Local structures in protein folding
    Undergo hydrophobic collapse to molten globule state
  103. Stabilization of secondary structure in protein folding
    Internal side chains pack together, water is expelled from protein core
  104. Image Upload 28
    • Folding funnel
    • Proteins fold via a series of conformational changes that reduce their free energy and entropy until the native state is reached
    • there are many paths to the bottom of the funnel
  105. Image Upload 29
    • Simplified smooth folding funnel
    • "single step" folding
  106. Image Upload 30
    • More realistic folding funnel
    • "multi-step" folding
  107. Normal protein folding pathway
    • Synthesis
    • Denatured (unfolded) 
    • Intermediate
    • Native
    • Fiber
  108. Image Upload 31
  109. Amyloid fibril
    • A misfolding that is tightly packed and therefore hard to degrade
    • Also does not take more energy to fold an amyloid than amorphous aggregate and will spontaneously form
  110. What are amyloids heavily enriched in?
    • Beta sheets which are very stable due to a large number of hydrogen bonds (often hydrophobic clusters)
    • Folding beta sheets is kinetically slower as the stabilizing H-bonds are separated by longer distances as compared to alpha helices (i-i+4)
  111. Alzheimers disease associated protein
    Amyloid beta or "Abeta" peptide
  112. Parkinson's disease
    alpha-Synuclein
  113. Spongiform encephalopathies (kuru)
    Prion protein
  114. Huntington's disease
    Huntingtin with polyQ tracts
  115. Molecular chaperones
    Large family of proteins that function to "inhibit inappropriate interactions between potentially complementary surfaces and disrupt unsuitable liasons so as to facilitate more favorable associations"

    Help to minimize aggregation and provide a framework for proper protein folding
  116. Chaperonin proteins
    • A particular class of chaperones
    • Aid in the proper folding of misfolded proteins with exposed hydrophobic patches
    • Some proteins cannot get folded without these
  117. How do chaperonin protein family work?
    • Initially exposed hydrophobic patches interact with an unfolded protein
    • ATP hydrolysis hides hydrophobic patches, exposing polar residues allowing folding and expelling of the bound protein
  118. Intrinsically disordered proteins (IDP)
    • Some proteins are natively unfolded i.e present completely or partially in a random coil form
    • The fraction of these increase from prokaryotes -> unicellular -> multicellular
  119. Where are IDPs found?
    • In "interaction hubs" and play a critical role in signaling and other processes when interaction with numerous partners is essential
    • Are highly interactive and can form complexes with a large variety of different proteins
  120. Tumor suppressor P53
    • An IDP that is a transcription factor and controls gene expression
    • Arrests cell cycle progression if the DNA is damaged
Author
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ID
348647
Card Set
Biochem Ch 4, 5, & 6
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Updated