Chapter 5 Protein Function

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Chapter 5 Protein Function
X-Ray crystal structure of insulin receptor and
inhibiting ligand
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Protein-ligand interactions case studyMyoglobin
(Mb)

• ? 16.7 kDa globular, 153 residue O2-binding
  protein
• Highly conserved in many organisms
• Found in high quantity in muscle
• Coordinated to Fe2
• Contains the heme prosthetic group
• Reversibly binds O2 for delivery to cells

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Myoglobin Structure and FunctionThe heme group
RED Porphyrin group
Hexa-coordinated iron ion (Fe2)

• Iron is in the Fe (II) oxidation state, which
  binds reversibly to molecular O2.
• Q. What prevents oxidation to Fe (III)?
• Electron-donating character of N atoms in
  porphyrin ring and
• Group is buried deep in interior of protein

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Fe is coordinated to 4 N atoms of heme, N of His
93, leaving one site for O binding
Fe2
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O2 Binding

• Binding requires access of O2 to the deeply
  buried heme
• Protein breathing allows transient openings to
  interior of protein
• Steric considerations affect binding of other
  molecules to heme (CO, CN-)

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Secondary and tertiary structure
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Protein-ligand equilibrium
Definition ? ? fraction of binding sites on
protein occupied by ligand Kd Ligand
concentration at which ½ of sites are
occupied (Half-saturation) NOTE Low Kd means
high protein affinity for ligand
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Myoglobin (P)-O2 (L) Binding

• For gases, use PRESSURE, so expression for ?
  changes to

where P50 is the partial pressure of O2 at half
saturation

• P50 of Mb 2.8 torr
• P(O2) 100 torr (arterial) and 30 torr (venous)
• Thus, Mb remains bound to O2 over wide range of P

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Hemoglobin (Hb)

• 4-subunit, 65 kDa protein, (4? structure)
  consisting of myoglobin-like units
• Hb is an allosteric protein (binding effects
  subsequent binding)
• 2 ? subunits 2 ? subunits
• Interactions between subunits at interface are
  hydrophobic, H-bonds, electrostatic
• 1 Hb binds 4 O2
• P nL ? PLn
• Hb-O2 dissociation curve is not described by
  hyperbola

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Mb vs. Hb
Mb binds O2 at pressures at which Hb releases it!
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Slope n, degree of Cooperativity n lt 1
Negative cooperativity n gt 1 Positive
cooperativity n 1 Non-cooperative
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Hb-O2 (OxyHB) Binding Mechanism

• T (tense) state Predominates in low O2
  environment (deoxyHB)
• Upon O2 binding, T ? R (relaxed) state (oxyHb)
• R state is stabilized by ligand (O2, CO, CN-)
  binding T state by salt bridges
• Conformation change is result of alterations in
  subunit interfacial contacts and interactions
• Changes in heme structure (pucker vs. planar)
  account for cooperativity and sigmoidal
  dissociation curve

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Transport of CO2 and HThe Bohr Effect
(Christian, Niels daddy)

• In periphery, PCO2 is high pH is low
• Low pH decreases Hbs affinity for O2
• CO2 H2O ? H HCO3- (catalyzed by carbonic
  anhydrase)
• Hb(O2)nHx O2 ? Hb(O2)n1 xH
• So, H and Hb-O2 affinity are inversely
  proportional
• CO2 binding also decreases Hb affinity for O2
• Protonation stabilizes T-state by affecting
  subunit interactions

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BPG

• Why does stripped Hb have a higher O2 affinity
  than Hb in whole blood?
• Negative charges interact via salt bridges to
  basic (positively charges) amino acids in Hb
• BPG decreases Hb-O2 affinity, allowing
  dissociation at lower pO2
• FHb (fetal Hb) has lower affinity for BPG than
  does AHb (adult) this means HIGHER O2 affinity
• High altitude acclimation involves production of
  BPG

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Immunology

• Highly specific interactions between protein (T
  cell receptors and antibodies) and antigens (any
  agent that elicits a cellular immune response
• Humoral system (fluid)
• Antibodies and immunoglobulins (Ig)
• Produced by B lymphocytes
• Bind and bacteria, viruses, foreign compounds
• Cellular system
• T lymphocytes (T cells cytotoxic T cells)
• Cells contain T cell receptors proteins on cell
  surface which recognize and bind antigens
• Helper T cells produce cytokines (e.g.,
  interleukins) which stimulate T cell
  proliferation

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How Not to Kill Yourself

• MHC proteins Distinguish between pathogens and
  self
• MHC Classes I and II
• Present protein fragments on cell surface
• Foreign peptides are recognized as such by T cell
  receptors and targeted for destruction
• T cells against self proteins are not allowed to
  mature (in thymus)
• Class II MHC proteins utilize helper T cells to
  stimulate response
• HIV targets these cells, significantly disturbing
  immune response

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Fab Antigen binding fragment Fc Base of Y
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Types of Antibodies

• IgA, IgD, IgE, IgG, IgM
• IgM is earliest present in immune response
• IgG is major in secondary response (memory B
  cells)
• Leukocytes are activated, which destroy invaders
  by phagocytosis

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Antibody-antigen interactions are the basis for
many analytical techniques

• Polyclonal antibodies Many B lymphocytes raised
  against 1 antigen
• Bind different parts of same antigen
• Monoclonal antibidies Raised in identical B
  cells against 1 antigen
• Whole population binds the same site on antigen
• Both can be used, e.g., for affinity
  chromatography
• ELISA (Enzyme-linked immunoabsorbant assay)
• Protein of interest linked to subphase
• Solution containing antibody is introduced binds
  (TIGHTLY) to protein
• Secondary antibody (raised against first
  antibody attached to enzyme that catalyzes
  color-producing reaction) is introduced
• Immunoblot assay
• Proteins are transferred from PAGE to membrane
  and washed with primary and secondary antibodies
• Linked enzyme catalyzes signal producing reaction
• See movie!

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Actin-Myosin SystemEnergy-requiring motility

• Based on protein-protein interactions
• Myosin (6-subunit protein thick filiment)
  contains several helices and ATP-binding domain
• Actin monomers aggregate to form thin filament
• Actin walks along myosin, resulting in muscle
  contraction and relaxation

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(Actin)
(Myosin)
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Muscle Contraction Regulation

• 2 proteins, troponin and tropomyosin, control
  actin-myosin muscle contraction
• Tropomyosin (in complex with troponin) binds
  actin, inhibiting actin-myosin interaction
• Ca, released as response to stimulus, binds
  troponin, causing conformational change, allowing
  actin-myosin interactions (contraction)