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 (>N-O)
• Atomic force microscopy measures heights of various
parts of specimen at the molecular level.
Suggestions for studying from your
fellow students (>90% 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 didn’t know as well. Then studied
mainly that material.”
• “… Re-wrote notes, made up questions and
answered.”
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 SDSPAGE, and apply your
knowledge to solve SDSPAGE problems.
• Describe the classes of
membrane proteins and
how they can be removed
from membranes.
A. SDS-PAGE
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.
SDS-PAGE
Reagents
• Polyacrylamide forms gel matrix
– small proteins go through fast
– large proteins go through slowly
• -mercaptoethanol breaks S-S bonds
HS-CH2-CH2-OH
– with: reducing conditions break
subunits apart
– without: non-reducing conditions keep
subunits together
– Allows determination of number of
subunits
B. RBC membrane proteins
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
Evidence for mosaic of proteins:
Freeze-fracture SEM
Freeze-Fracture SEM of membranes
< Artificial bilayer w/o protein
Artificial bilyaer w/protein >
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
Solubilization of integral membrane
protein by nonionic detergent
Critical micelle concentration
SDS-PAGE Problem
•
You are given a preparation of kangaroo membranes (M), part of which looks like this
(assume membrane is sealed):
Protein B
Protease
Out
membrane
In
•
Protein A
You do the following experiment, where an arrow indicates centrifugation:
Isolated
membranes
Sup S1
Add non-ionic
Sup S2
Salt
detergent
Spin
(M)
Pel
P1
wash
(to solubilize Spin
Pel P2
membranes)
• 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.
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:
M
M
•
S1
S1
P1
P1
S2
S2
P2
P2
PRO
PRO
Homework: Draw a diagram of what the aardvark membrane looks like
(Hint: the protein may cross the membrane more than once).
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.
Example of connexin: 4 positive peaks
from hydropathy analysis predicts the
protein has 4 transmembrane domains.
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
Glycophorin and Bacteriorhodopsin
• Bacteriorhodopsin was one of first membrane proteins whose 3D
structure was determined.
– It functions as a light-driven proton pump
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.
N-linked and O-linked glycosylation
• Linkage to either N or O
on R groups
• Function of glycoproteins:
usually in plasma
membranes, role in cellcell recognition along with
membrane receptors
• Glycosylation occurs in
ER and Golgi
Chains vary from 2-60 sugar units
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