Announcements, Feb' 12

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Announcements, Feb. 12

• Happy Darwin Day! (b. Feb. 12, 1809)
• Reading for today 172-186 on membrane proteins.
• Reading for Wednesday 191-207 on membrane
  transport.
• SDS-PAGE homework problem due
• Also on Wednesday Representatives in biology
  will be at the Annual Summer Job Fair, UC Rotunda
  and Terrace Rooms, from 11 AM to 4 PM.
• Reading for Friday 207-216 on energetics of
  membrane transport.
• Electron spin resonance (ESR) spectroscopy
  measures lipid mobility in membranes
• Similar to NMR, labeling with nitroxyl group
  (gtN-O)
• Atomic force microscopy measures heights of
  various parts of specimen at the molecular level.

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Suggestions for studying from your fellow
students (gt90 on Exam 1)

• I went to the study session, had a study group,
  and read over the powerpoints.
• I read through the powerpoint slides.
• I started studying 5 days before studied 2
  sets of notes each night then all on the
  Thursday night before the test. I also looked at
  diagrams on the power points.
• I rewrote my notes and made notes from that on
  the stuff I didnt know as well. Then studied
  mainly that material.
• Re-wrote notes, made up questions and
  answered.

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Outline/Learning Objectives

• Membrane Proteins
• SDS-PAGE
• Membrane proteins in red blood cells
• Classes of MB proteins
• Orientation and glycosylation of MB proteins

• After reading the text, attending lecture, and
  reviewing lecture notes, you should be able to
• Explain how proteins can be separated by
  SDS-PAGE, and apply your knowledge to solve
  SDS-PAGE problems.
• Describe the classes of membrane proteins and how
  they can be removed from membranes.

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A. SDS-PAGE
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SDS-PAGE Reagents

• Sodium dodecyl sulfate
• Strong ionic detergent
• Removes proteins from MB, solubilizes hydrophobic
  amino acids
• Unfolds and coats proteins w/ negative charge
• Triton X-100
• Mild non-ionic detergent
• Removes proteins from MB
• Solubilizes proteins but does not unfold them.

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SDS-PAGE Reagents

• Polyacrylamide forms gel matrix
• small proteins go through fast
• large proteins go through slowly
• ?-mercaptoethanol breaks S-S bonds
• with reducing conditions break subunits apart
• without non-reducing conditions keep subunits
  together
• Allows determination of number of subunits

HS-CH2-CH2-OH
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B. RBC membrane proteins
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Functions of MB Proteins

• Receptors or signals, e.g. glycophorin
• Structural, e.g. spectrin, ankyrin, Band 4.1
• Transporters, e.g. glucose transporter, Band 3
• Channels, e.g. Na channels in excitable cells
• Enzymes, e.g. G3PDH
• Electron transport proteins in mitochondria,
  chloroplasts
• Intercellular adhesion and communication, e.g.
  gap junctions

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Evidence for mosaic of proteins Freeze-fracture
SEM
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Freeze-Fracture SEM of membranes
lt Artificial bilayer w/o protein Artificial
bilyaer w/protein gt
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C. Classes of Membrane Proteins
transmembrane
1. Integral membrane proteins require detergent
to remove from MB 2. Peripheral membrane
proteins removed by milder treatments 3.
Lipid-anchored membrane proteins in lipid rafts
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Solubilization of integral membrane protein by
nonionic detergent
Critical micelle concentration
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SDS-PAGE Problem

• You are given a preparation of kangaroo membranes
  (M), part of which looks like this (assume
  membrane is sealed)
• You do the following experiment, where an arrow
  indicates centrifugation
• You also treat M with protease, on the side of
  the bilayer indicated in the diagram. This
  sample is called PRO.
• All samples (M, S1, P1, S2, P2, PRO) are mixed
  with SDS and run on a denaturing polyacrylamide
  gel. Diagram what you expect to see in the gel
  for each sample.

Protease
Protein B
Out In
membrane
Protein A
Isolated membranes (M)
Sup S1 Pel P1
Add non-ionic detergent (to solubilize membranes)
Sup S2 Pel P2
Salt wash
Spin
Spin
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Solution and Homework

• Part 1
• Part 2 You then get adventurous, and look at
  the membrane from an aardvark cell, repeating the
  exact same protocols as for the kangaroo
  membranes. You also run a gel, as above, and see
  this
• Homework Draw a diagram of what the aardvark
  membrane looks like (Hint the protein may cross
  the membrane more than once).

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1. Integral Membrane Proteins

• Strong treatments (detergents) are required to
  remove from MB.
• Amphipathic molecules
• Transmembrane regions are ? -helical with
  hydrophobic R groups facing out - usually 20-30
  amino acids.

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2. Peripheral and 3. Lipid-Anchored Membrane
proteins

• Peripheral MB proteins
• Weak treatments (change in pH or ionic strength,
  removal of Ca2) remove from MB since bound by
  electrostatic interactions or H-bonds.
• Can be on outside or inside spectrin, ankyrin,
  and Band 4.1 are inside examples from RBCs.

• Lipid-anchored MB proteins
• Covalently bound to membrane lipids.
• Most bound to fatty acids on inner leaflet.
• Some bound to outer leaflet linked to GPI (a
  glycolipid in external monolayer)
• May be enriched in lipid rafts

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Glycophorin and Bacteriorhodopsin

• Bacteriorhodopsin was one of first membrane
  proteins whose 3D structure was determined.
• It functions as a light-driven proton pump

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D. Many membrane proteins are glycosylated

• In addition to lipids and proteins, most
  membranes have significant amounts of
  carbohydrates
• Erythrocyte - 52 protein, 40 lipid, 8 carb.
• Glycolipids account for only small portion of
  membrane carbohydrates most is in form of
  glycoproteins.
• Addition of carbohydrate side chain to a protein
  is glycosylation.

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N-linked and O-linked glycosylation

• Linkage to either N or O on R groups
• Function of glycoproteins usually in plasma
  membranes, role in cell-cell recognition along
  with membrane receptors
• Glycosylation occurs in ER and Golgi

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Chains vary from 2-60 sugar units
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Purpose of protein glycosylation

• Synthesis of complex carbohydrates requires a
  separate enzyme for each different step, unlike
  other polymerization reactions.
• May be several functions, not well-understood
• Presence of oligosaccharides makes glycoprotein
  more resistant to digestion by extracellular
  proteases.
• Glycosylation also may be important for
  receptor-ligand binding.
• CHO-binding proteins are called lectins