I.
II.
III.
Many functions of a membrane are reflected in the types and amounts of its
proteins.
a. Myelin
i. ~25% protein by mass
b. Inner membrane of a mitochondria
i. ~75% protein by mass; energy transduction reactions
c. Typical Plasma membrane
i. ~50% protein by mass
1. there are 50x more lipid molecules than protein molecules
Protein function is determined by the nature of the protein-membrane
association
a. Classification of membrane-associated proteins
i. Integral/Transmembrane Proteins
1. Penetrate the lipid bilayer
a. Domains protrude both sides
i. Extracellular
ii. Cytoplasmic
2. Types
a. Single pass
i. Pass through the membrane once
b. Multi pass
i. Goes back and forth across membrane
3. Make up 20-30% of all encoded proteins
ii. Peripheral membrane proteins
1. Entirely outside of the lipid bilayer
a. Cytoplasmic or Extracellular side
2. Associated to the surface of the membrane by noncovalent
bonds
a. Ionic bonds
i. Sodium will break ionic bonds
b. Hydrogen bonds
i. Urea will break hydrogen bonds
iii. Lipid-linked Proteins
1. Lie on the outside of the lipid bilayer
a. Cytoplasmic or Extracellular side
2. Covalently linked to a lipid molecule that is situated within the
bilayer.
a. Enter the membrane by being associated with a fatty
acid
Detergents are used to solubilize membrane proteins
a. Non-ionic
i. Triton X-100
1. Mild
2. Ether linkage
b. Ionic
i. SDS
1. Denatures
a. Sulfate group at the end
i. Negative charge
b. Sodium ion usually associated with it
c. Amount of detergent used is analogous to protein size
IV.
V.
d. Both are Amphipathic
i. Detergent’s hydrophobic tails towards hydrophobic center of protein
1. Detergent solubilizes hydrophobic portion of protein
a. Allows for removal
Integral Membrane Proteins Revisited
a. Identification of IMPS
i. They are Amphipathic
ii. Hydropathy plots
1. X-Axis
a. Amino Acid #
2. Y-Axis
a. Delta G (Free energy)
3. Calculates the amount of free energy needed to transfer
successive amino acid segments from non-polar solvent to
water.
a. Regions of protein containing 20-30 amino acids that
showed a positive (high) G are most likely membrane
spanning regions
i. They are non-polar
1. Not too happy about the transition to
water.
b. Characteristics of membrane-spanning regions
i. -Helix
1.
ii. -barrel
1. Series of  second
2. Creates a pour through the outer membrane of the
mitochondrion
a. Porin molecule
i. In bacteria outer membrane
iii. Similarities
1. Belt of aliphatic (not aromatic) amino acid side chains
a. Located in hydrophobic region
2. Flanked by two aromatic girdles
Topography of membrane proteins
a. Determining Topography (orientation) of the protein
i. Vectorial Labeling
1. Tag portion of protein with radioactivity/fluorescence
a. Called reporter
2. Use probe itself that cannot cross the membrane
a. Labeling outside
i. Not in hypotonic medium
b. Labeling outside and inside
i. Put in hypotonic medium then isotonic
1. Cause cell to disassemble and reassemble
a. Can get probe on the inside
ii. Proteolytic enzymes
1. Add trypsin to
a. Intact cell
b. Permeabilized cell
VI.
2. Eliminates the protein domains on the outer surface of the cell
and on both sides of the Permeabilized cell.
a. Membrane fluidity increase allows crossing of
membrane
iii. SDS-PAGE
1. Protein with 2 subunits joined by a disulfide bridge
2. Single subunit protein
3. Treat both proteins with SDS and mercaptoethanol
a. Denatures protein
b. Negatively charged
4. Run gel electrophoresis
a. Start at negative cathode and will run towards positive
anode.
5. Lighter proteins will travel further.
a. See lecture handout page 3
Movement of membrane proteins
a. Proteins can diffuse rapidly in the plane of the membrane
i. Frye and Edidin experiment
1. First direct evidence of lateral diffusion of proteins
2. Fused a mouse and human cell together using fusing virus
3. Antigens of each cell dyed a different color
a. After 40 mins
i. Even distribution of proteins
ii. Rates of diffusion can be determined by FRAP
1. Label proteins with fluorescent dye
2. Photobleach spot with laser beam
a. Record the rate and to the extent other proteins in the
cell fill the hole.
b. Many membrane proteins (and some lipids) have restricted membrane
mobility.
i. Cellular mechanisms for restricting the lateral mobility of proteins
1. Tethered to cell cortex on inside
a. Actin
i. Protein in the cytoskeleton
1. Plays part in tethering proteins to cell
b. Network of fences in underlying skeleton
c. Keep the proteins close to facilitate interaction
d. Disassembles and reassembles periodically
2. Extracellular matrix molecules
a. Web of materials prevents proteins (not connected to
inside of cell) from leaving the membrane
b. Collagen is a common protein used to connect the
proteins
3. Proteins on surface of adjacent cell
a. Adjacent membrane crossing domains
b. Integrins
i. Proteins on the outside of the cell that reach out
and hook integrins on neighboring cells
1. In a Velcro like fashion
4. Diffusion Barriers
a. Also called tight junctions
b. Optical tweezers
c. Elastic barrier
d. Drag for a while and then stops and then shoots back