Membrane Proteins

1
Membrane Proteins

• Mosaic part is the proteins
• Found through Freeze Fracture technique break
  in the path of least resistance
• Can see proteins on the inner P face
  (protoplasmic, inner leaflet) and on the outer E
  face (exoplasmic, outer leaflet)

2
Different Freeze Fractures
3
Protein Types

• Proteins differ in their affinity for the
  hydrophobic layer and therefore dictates the
  interaction with the membrane

4
Integral Membrane Proteins

• Possess 1 or more hydrophobic regions in the
  primary sequence that embed within the membrane,
  will also have hydrophilic region that have
  contact with 1 or both membrane surfaces
• Most or all of the amino acids passing thru the
  membrane have hydrophobic R groups
• Hard to remove from the membrane without
  detergents

5
Types of Integral Proteins

• Monotopic embedded in only 1 leaf of membrane,
  inner or outer
• Transmembrane pass through the membrane
• Most of the proteins
• 1 pass through single-pass
• 2 or more passes through multipass 2-20 or
  more passes
• Multipass proteins may have more than 1 subunits
• All are anchored in the membrane by the
  hydrophobic R groups, usually an ?-helix but
  occasionally ?-sheet making a ?-barrel (porin
  proteins)
• Single pass may have C or N terminus on
  different sides or on the same side of the
  membrane

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RBC Membrane

• Glycophorin is a single pass protein with N
  terminus on the outside
• Band 3 Protein is a multipass protein with the C
  and N on the same side of membrane
• 2 subunit protein that spans the membrane 6 times

7
Bacteriorhodopsin

• Proton pump in purple bacteria
• Forms a channel that moves protons across the
  membrane
• 7 ? helices with 20 hydrophobic AA

8
Peripheral Membrane Proteins

• Do not penetrate the membrane
• Spectrin, ankyrin and band 4.1 are examples from
  the RBC membrane, helps maintain the shape of the
  cell, supports membrane on the inner surface
• Bond to the surface of the membrane by weak
  electrostatic forces and H-bonding with the polar
  heads or hydrophilic integral proteins
• Extract by changing the pH or ionic strength

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Lipid-Anchored Membrane Proteins

• Have characteristics of both of the previous
  types
• Protein sits on the membrane but it is covalently
  linked to a lipid molecule that is embedded in
  the membrane
• Anchored by a fatty acid, prenyl group or
  glycosylphosphatidylinositol (GPI)
• Fatty acid and prenyl groups anchor to the inner
  leaflet
• GPI anchors to the outer leaflet
• Easily removed but must remove the lipid portion

10
Lipid Anchored Proteins

• Attached to fatty acid protein synthesized in
  cytosol and the covalently linked to FA in
  membrane
• Attached to prenyl group soluble cytosolic
  protein before addition of prenyl group and then
  inserted into membrane
• Attached to glycosylphosphatidylinositol (GPI)
  GPI is a glycolipid that has a single pass
  protein attached and then the transmembrane
  portion is cleaved off

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Protein Separation
Move inversely related to size
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Molecular Biology

• Electrophoresis uses electricity to separate
  proteins based on size and shape
• SDS (sodium dodecylsulfate) coats the protein and
  unfolds it as well as giving it a negative charge
• Disturbs protein/protein and protein/lipid
  interactions
• Proteins move through gel to positive electrode
  based on size smaller move faster and move
  farther
• Recombinant DNA technology is useful to study
  function and what happens when AA are changed
  (Box 7-A, should be familiar with this)
• X-ray crystallography gets the 3D structure of
  a protein

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Hydropathy Plots Computer Algorithm

• Used to predict the transmembrane spanning
  regions in the amino acid sequence
• Creates an hydropathy index positive peaks
  indicate the transmembrane area

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Variety of Functions

• Enzymes specific functions to specific
  membranes
• Electron transport proteins function in
  oxidative phosphorylation
• Transport proteins move nutrients
• Channel proteins hydrophilic molecule passage
• Transport ATPase move ions by use of ATP
• Receptors recognize and impinge on cell surface
  and transmit thru cell
• Intercellular communications gap junctions and
  plasmodesmata
• Structural proteins Stabilize membrane and cell
• Additional ones important to the cells function

15
Asymmetric Orientation

• With respect to the bilayer and its peripheral,
  lipid-anchored and monotopic proteins
• All the same protein are located on the same side
• Have specific membrane orientation
• The part of the protein that is exposed on one
  side of the membrane is different than the other
  side
• Membranes are also functionally asymmetric
  reactions that occur on one side are different
  than the other surface

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Determination of Protein Location

• Can label the proteins on either side of the
  membrane depending on where the reagents are
• Antibodies are also useful to determine location

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Glycoproteins

• Proteins are glycosylated sugar added to the
  protein AA side chains glycosylation
• On the external surface of the membrane
• Function in cell to cell recognition

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Glycocalyx

• Glycoproteins and glycolipids on the surface of
  the cell
• Makes a very moist layer because of the
  hydrophilic nature of the sugars and its
  interaction with water

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Glycosylation

• N-linked sugar added to the N in the amino
  group asparagine
• O-linked sugar added to an O in the hydroxyl
  group usually serine, threonine, hydroxylysine
  or hydroxyproline
• Sugars are either branched or straight
• Sugars can be identified by using lectins
• Either at the end of the chain or in the middle
  of the sequence

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Common Sugars
Helps RBCs repel each other and help with blood
flow and RBC removal
21
Protein Mobility Cell Fusion
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Protein Movement

• Membrane proteins will move at more variable
  rates than lipids due to their size
• Protein movement may be restricted by the
  presence of barriers such as the tight junction
  that connect cells or by the protein being
  anchored to a structure in the cell
• Polarized cells have proteins restricted to a
  specific part of cell membrane