Biological

1
Chapter 10

• Biological
• Membranes

2
What you should understand

• Lipid-protein associations
• The pathway for protein insertion in membranes
• Specific membrane functions including mediated
  and active transport systems

3
Membrane Roles

• Compartmentalization
• Permeability barrier
• Transport
• Allow transfer of selected molecules
• Signaling
• Surface receptor recognition and signal transfer

4
Membrane Components

• Membranes Proteins Lipids
• Ratios vary from 0.23 to 3.2
• Eukaryotic average to be 50 (11 ratio)
• Classified according to membrane association as
• INTEGRAL or PERIPHERAL

5
INTEGRAL or INTRINSIC Proteins

• Tightly bound by hydrophobic interactions
• Separated only by membrane disruption
• Organic solvents (ether)
• Detergents (SDS)
• Chaotropic Agents (Guanidinium Ion)
• Precipitate in aqueous solution unless
  solubilized by these agents

6
INTEGRAL or INTRINSIC Proteins

• Asymmetrically Bound Amphiphiles
• Surface labeling has demonstrated the residues
  that are not buried in the membrane
• Antibody recognition and binding
• Fluorescent labeling
• Radioactive labeling
• Protease digestion

7
INTEGRAL or INTRINSIC Proteins

• Asymmetrically Bound Amphiphiles
• Proteins have been shown to be both transmembrane
  or associated with only one leaflet or the other
• No proteins have been identified which are
  completely buried (?)
• ?-helices or antiparallel ?-sheet barrels are
  common membrane-spanning structures

8
INTEGRAL or INTRINSIC Proteins

• Transmembrane and exposed domains can be
  predicted by
• Hydropathy index for local sequence
• Free energy predictions for hydrophilic to
  hydrophobic transfer

9
INTEGRAL or INTRINSIC Proteins
GLYCOPHORIN A - 131 residue transmembrane protein
H3N-
Outside
30 Å
Inside
-COO-
O-linked (Ser, Thr) oligosaccharides N-linked
(Asn) oligosaccharides
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INTEGRAL or INTRINSIC Proteins
Gram Bacterial Porins - 16-18 stranded AP
barrel
H3N-
Outside
Inside
-COO-
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INTEGRAL or INTRINSIC Proteins

• Other Examples
• Bacteriorhodopsin seven 25 a.a. transmembrane
  helices with light-sensitive retinal
  residuelight-sensitive proton pump for ATP
  synthesis
• Photosynthetic Reaction Center eleven helices
  hydrophobicities of the membrane-spanning helices
  are amphiphilic purple bacteria

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INTEGRAL or INTRINSIC Proteins
Bacteriorhodopsin
Outside
Inside
Hydrophilic
Hydrophobic
13
Lipid-Linked Proteins

• Pyrenylated
• Fatty Acylated
• Glycosylphosphatidylinositol-linked
• May contain more than one
• Anchor protein in membrane

14
Lipid-Linked Proteins

• Pyrenylated Proteins
• Isoprene-based lipid
• Farnesyl (C15) or Geranylgeranyl (C20)
• Usually attached at C-terminal tetrapeptide
  recognition sequence C-X-X-Y

CH3 CH2 - C CH CH2
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Lipid-Linked Proteins

• Fatty Acylated Proteins
• Myristoylation via amide bond at N-terminal Gly
• Palmitoylation via thioester bond at Cys
  reversible by thioesterase for signal events

16
Lipid-Linked Proteins

• Glycosylphosphatidylinositol Proteins
• Phosphatidylinositol core serves as membrane
  anchor
• Glycosidically linked to tetrasaccharide to
  protein C-terminus (outer surface not generally
  transmembrane proteins)

17
Peripheral Membrane Proteins

• Peripheral or Extrinsic Proteins
• Do NOT bind lipid
• Dissociated by mild methods pH or ionic
  strength changes
• Bind to integral membrane proteins by
  electrostatic or H-bonds
• Example Cytochrome C

18
Membrane Structure Assembly

• Freeze-fracture electron microscopy interior
  of leaflets
• Freeze-etching surfaces
• Demonstrates asymmetry
• Rough membrane interior
• Smooth membrane exterior

19
Membrane Structure Assembly

• The Erythrocyte Membrane
• Membrane ghosts demonstrate a semi-rigid
  cellular skeleton
• Major unit spectrin, an (ab)2 structure
• (antiparallel ab diamer in head-to-head diamer)
• Actin, Tropomyosin, and Ankyrin serve as
  co-anchoring units (Fig 10-15)

20
Lipid Asymmetry

• Lipid Synthesis and transport Facilitated
  Diffusion - Flipases

TNBS - immediately
Outer
TNBS after 3 min. delay
32PO4-2
Inner
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Lipid Asymmetry

• Lipid Synthesis and transport
• 1. Facilitated Diffusion Flipases
• From higher to lower concentration
• 2. Active Transport Phospholipid translocases
• ATP-driven
• 3. Membranes as synthesized from other
  membranesall are asymmetric

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Lipid Asymmetry
Human Erythrocyte Membrane Lipid Distribution
Inner Leaflet
Outer Leaflet
Total Phospholipid
Sphingomyelin ( -)
Phosphatidylcholine ( -)
Phosphatidylethanolamine ( -)
Phosphatidylserine ( - -)
0
50
50
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The Secretory Pathway

• Protein synthesized N to C on free or bound
  ribosomes
• Free cytoplasmic and mitochondrial
• Bound transmembrane, secreted, ER or
  lysosomal
• Bound synthesis via Signal Hypothesis

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The Secretory Pathway

• Signal Hypothesis
• Secreted, ER, transmembrane and lysosomal
  proteins have 13-36 signal peptide at N-terminus
• Once the signal protrudes from the ribosomal
  surface, SRP (signal recognition peptide) stops
  translation and diffuses complex to the RER to
  the SRP Receptor (docking protein)

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PROTEIN SYNTHESIS
DNA
Transcription
mRNA
Translation
Globular Protein
Ribosome attaches to ER
Amino Acids
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The Secretory Pathway

• Signal Hypothesis
• Docking reinitiates translation
• GTP binds and the protein is guided through the
  RER transmembrane channel
• GTP hydrolyses and signal peptidase cleaves the
  signal from the preprotein
• Chaperones and PDI facilitate folding in the
  lumen of the ER
• Post-translational modifications begin

27
The Secretory Pathway

• Signal Hypothesis
• Translation finishes and ribosome dissociates
• Transmembrane proteins remain membrane-anchored
  (20 a.a. sequence) with C-terminus on the
  cytoplasmic side of ER
• Others float free in ER lumen

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The Secretory Pathway

• Signal Hypothesis
• Applied to both Eukaryotes and Prokaryotes
• Does not account for ALL transmembrane transport
• Chaperone proteins provide an alternate process
  of membrane transport

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The Secretory Pathway

• Targeting Specific Destinations Budding Coated
  Vesicles
• RER to Golgi - COPI and COPII proteins fuzzy
  vesicles
• Golgi to Membranes Clathrin polyhedral
  vesicles
• Budding always conserves protein-membrane
  orientation lumen outside of cell
  glycosylated and/or N-terminus
• Soluble ER proteins are retrieved by KDEL
  sequence

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Lipoproteins (circulating lipids)

• Lipoproteins globular, micelle-like particles
• Nonpolar core of TAGs and Cholesterol esters
  surrounded by amphiphilic protein, phospholipid,
  cholesterol coat
• Apolipoproteins A-1 (243 residues) and B-100
  (4536 residues) are typical associated proteins
  that help to solubilized the lipid micelle
  through amphiphilic helical structures. (Note
  Helical Wheel Diagrams, Figure 10-26)

31
Lipoproteins

• Lipoproteins 5 Classes (Table 10-1, page 261)
• 1. Chylomicrons exogenous TAGs and cholesterol
  from intestines to tissues (least dense, largest
  particle)
• 2. VLDL , IDL, LDL endogenous TAGs and
  cholesterol from liver to tissues
• LDL uptake is receptor mediated
• 3. HDL endogenous cholesterol from tissues to
  liver (most dense, smallest particle)

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Lipoproteins

• Cholesterol Uptake by the LDL
• Receptor-mediated uptake via clathrin-coated pits
  on membrane border
• LDL is internalized by endocytosis and fuses with
  endosome
• LDL receptors and clathrin are separated
  receptor return to membrane by exocytosis,
  clathrin by diffusion
• LDL-endosome fuses with lysosome for digestion to
  amino acids, cholesterol and fatty acids

33
Lipoproteins

• Cholesterol Uptake
• Familial Hypercholesterolemia is a genetic-based
  deficiency of LDL receptors
• This leads to high plasma (extracellular) levels
  of cholesterol and, hence, deposit in the
  arteries and veins

34
Transport Across Membranes

• Water ? Lipid ? Water
• Non-Mediated Transport (Diffusion)
• Mediated Transport
• Passive-mediated (Facilitated) Transport
  movement from High ? Low
• Active Transport
• movement from Low ? High
• Can we predict which occurs?
• Ask What is the energy cost?

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Transport Thermodynamics

• Water ? Lipid ? Water
• How do we know the energy cost?
• Ask What is the chemical potential difference?
• ?G -RT ln Keq (Chapter 1)
• Modified for free energy change across a
  membrane
• ?GA RT ln Ain ZA F ??
• Aout

( )
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Transport Thermodynamics

• Water ? Lipid ? Water
• ?GA RT ln Ain ZA F ??
• Aout
• Where the ?A and charge on the ion (Z) become
  the controlling variables

( )
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Transport Thermodynamics

• Water ? Lipid ? Water
• So
• Transport requires energy when
• going from low A to high A (against the
  gradient), and/or
• an ion is highly charged (Z) poorly soluble in
  the membrane

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Transport

• Water ? Lipid ? Water
• Non-Mediated Transport (Diffusion)
• Flow is in the direction to eliminate the
  concentration gradient
• Rate depends on the solubility of the molecule in
  the membrane

39
Transport

• Water ? Lipid ? Water
• Non-Mediated Transport (Diffusion)
• Although polar, H2O can diffuse across
  membranes despite the charge due to
• Concentration Gradient
• Small size

40
Transport

• Water ? Lipid ? Water
• Mediated Transport
• Passive-mediated transport
• High ? Low (large, polar molecules traveling
  WITH gradient)
• Active transport
• Low ? High (endergonic-traveling AGAINST the
  gradient)

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Transport

• Mediated Transport molecules too large, too
  polar, moving against gradient
• Utilize carriers, permeases, porters,
  translocases, transporters

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Receptor-Mediated Transport
Cells without mediated transport
Cells with Na-coupled mediated transport
Rate of Glucose Uptake
Mediated transport with 6-O-benzyl-D-galactose
Na
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Transport

• Passive - Mediated Transport
• A. Ionophores (Amphiphilic Compounds)
• Carrier valinomycin K specific carrier,
  disrupts Na/K pump gradient transporting 104
  ions/sec
• Channel gramicidin A K carrier, disrupts
  Na/K pump gradient transporting 107 ions/sec

44
Transport

• Water ? Lipid ? Water
• Passive -Mediated Transport
• B. Porins Forms a constricted channel with
  mid-pore lysine facilitating anion passage

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Transport

• 2. Passive - Mediated Transport
• Transport Proteins
• Glucose Transporter Protein
• Glucose binds protein open to outer side of
  membrane
• Conformational change closes binding site at
  membrane outer side and opens site at inner side
• Glucose dissociates to inside
• Protein returns to initial conformation (open to
  outer side)

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Transport

• 2. Passive - Mediated Transport
• Transport Proteins
• Glucose Transporter Protein
• Can transfer either direction depending on
  gradient
• Asymmetric, allosteric conformational changes
  (like hemoglobin)

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Transport

• 2. Passive (AND Active) - Mediated Transport
• Transport Proteins
• Can pair transport of molecules
• Uniport
• Symport
• Antiport

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PASSIVE MEDIATED (Facilitated) TRANSPORT
A
B
A
A
Outside
Inside
B
Uniport
Symport
Antiport
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ACTIVE TRANSPORT
(Na K ) ATPase
8-helical segments 2 large cytoplasmic domains
Outside
?
?
?
?
Inside
3 Na (in) 2K (out) ATP H2O ? 3 Na (out)
2K (in) ADP Pi
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ACTIVE TRANSPORT
(Na K ) ATPase (Antiport-maintains osmotic
gradient)
Outside
?
?
ATPase Asp binds ATP with Na hydrolyzes with
K
?
?
Inside
3 Na (in) 2K (out) ATP H2O ? 3 Na (out)
2K (in) ADP Pi
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(Na K ) ATPase
H2O
2 K (out)
Pi
Outside
E2P
3 Na (out)
E22K
E2P2K
2 K (in) 3 Na (in)
E13Na
E1ATP3Na
E1P3Na
Inside
ATP / Mg2
ADP / Mg2
1.
3 Na (in) 2K (out) ATP H2O ? 3 Na (out)
2K (in) ADP Pi
52
Ca2 ATPase

• Active Transport - Ca2 is four orders of
  magnitude greater outside the the cell
• Ca2 is pumped OUT of the cells with the help
  of ATP
• Similar mechanisms are used by ER, plasma
  membrane, and sarcoplasmic membrane of muscles

53
H -K ATPase

• ACTIVE TRANSPORT - Maintains 6-unit pH gradient
  between the cytosol of cells of the stomach (pH
  7) and stomach contents (pH 0/8)
• Activated by histamine blocked by cimetidine
• Histamine release induced by H. pylori infection

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Ion Gradient-Driven Transport

• Active transport systems can generate
  electrochemical gradients across membranes
• This gradient can provide energy for other
  transport systems via Secondary Active
  Transport
• EXAMPLE 1 Na-Glucose Transport System keeps
  internal Na low so that Na-Glucose symport
  from the intestinal lumen is favored by a
  concentration gradient. Uniport then dumps
  glucose from the intestinal cells to the
  capillaries.

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Ion Gradient-Driven Transport
Secondary Active Transport EXAMPLE 2 Lactose
Permease in E. coli uses the proton gradient
from oxidative metabolism to provide energy to
transport lactose into the cell
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Lab Positions

• Melittin Purification
• Hemolysis Assay
• Antibacterial Assay
• Gel Electrophoresis
• Protein Assay