Download Membrane Proteins

BIOC 460, Summer 2010
Membrane Proteins
Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 12, pp. 336-348
Bacteriorhodopsin Jmol structure:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/rhodopsin/rhodop1.htm
PGH2 Synthase (COX 2) Jmol structure:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/cox12/cox121.htm
Bacterial porin Jmol structure:
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/porin/newporin.html
Dynamics of membrane lipids as well as the proteins embedded with the lipid bilayer
and is highly recommended:
http://multimedia.mcb.harvard.edu/anim_innerlife.html
Key Concepts
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Membrane Proteins
Membrane functions (review): selective permeability barriers, information
processing, organization of reaction sequences, energy conversion
Lipids (lipid bilayer) responsible for permeability barrier
Proteins perform essentially all other membrane functions, including
modulation of permeability barrier by allowing or assisting some solutes to
cross membrane (transport processes)
Fluid mosaic model of membrane structure: 2-dimensional "fluid"
composed of lipids and proteins (both often with attached carbohydrates
on outer side of membrane)
Proteins: peripheral, lipid-anchored, or integral
Mobility of components within the membrane:
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Lateral diffusion: rapid for both proteins and lipids within the plane
of the membrane (except for proteins anchored, for example to
cytoskeleton)
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Transverse diffusion ("flip-flop") of both proteins and lipids is
extremely slow, unless mediated by protein "flippases".
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Lipid composition in the 2 leaflets of bilayer is asymmetric, as is
protein distribution.
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BIOC 460, Summer 2010
Key Concepts, continued
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Membrane fluidity essential to function, regulated by fatty acid
composition of lipids and (eukaryotes) by cholesterol content
Integral membrane proteins typically assume one of two secondary
structures to get polar groups of polypeptide backbone across
hydrophobic core of lipid bilayer: membrane-spanning 〈 helices (about 20
residues long) or antiparallel ® sheets wrapped into ® barrels.
Examples:
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Glycophorin: single hydrophobic transmembrane 〈 helix
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Bacteriorhodopsin: 7 transmembrane helices
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Prostaglandin H2 Synthase: on surface of ER membrane but
anchored in membrane by a set of 〈-helices with hydrophobic R groups
that extend into membrane core
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Porins: large ® barrel with aqueous channel down the center
Membrane Proteins
• mediate nearly all membrane functions except establishment of
permeability barrier
• membrane protein functions:
– "pumps" (active transport)
– "gates" ("passive" transport, facilitated diffusion)
– receptors
– signal
i
l ttransduction
d ti
– enzymes
– energy transduction
• Membrane protein distribution:
– Both amount of protein in general and which specific proteins are
present varies with function of membrane, i.e., with type of
membrane and with cell type.
– Examples:
• myelin (membrane around myelinated nerve fibers,
function=electrical insulation) mostly lipid (only ~18% protein)
• plasma membrane: enzymes, receptors, etc. (~50% protein)
• mitochondrial inner membrane and chloroplast thylakoid
membrane: electron transport, energy transduction (ATP
synthesis) (~75% protein)
Membrane Proteins
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BIOC 460, Summer 2010
Schematic Diagram of Membrane Structure
Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 11-3
Fluid Mosaic Model of Membrane Structure
(Singer and Nicholson, 1972)
• A 2-dimensional "solution" of globular proteins embedded in fluid lipid
bilayer, structurally and functionally asymmetric with respect to the 2 sides
of bilayer
• Proteins and lipids: free lateral diffusion (in plane of membrane).
• Transverse diffusion ("flip-flop") is very slow (thermodynamic and kinetic
barrier) without a catalyst
catalyst.
• Carbohydrates
on both proteins
(glycoproteins)
and lipids
(glycolipids)
are exposed on
extracellular
surface of
plasma
membrane.
Nelson & Cox, Lehninger
Principles of Biochemistry,
4th ed., Fig. 11-3
Membrane Proteins
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BIOC 460, Summer 2010
Membrane Asymmetry: Lipids
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Biological membranes are
asymmetric with respect to lipids
Lipid asymmetry is related to
function:
Plasma membrane: PS required in
outer leaflet for platelet formation
of blood clot
Other cells: PS in outer leaflet
signals for apoptosis.
Cardiolipin: (not shown) only found
in mitochondrial membranes
Membrane Asymmetry: Proteins
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Membrane Proteins
Asymmetric orientation of
proteins in planar membrane.
Protein orientation: Proteins
definitely oriented: inner side, or
outer side
side, or spanning
membrane, but in a specific
orientation
Orientation established during
proteins synthesis in ER
Orientation/structure DIRECTLY
linked to function: cell
recognition, signaling into cell,
transport of ions (H+, Na+, K+,
sugars) in or out of cell.
Oft structures
Often
t t
can be
b quite
it
elaborate: the mitochondrial
cytochrome bc1 complex.
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BIOC 460, Summer 2010
Membrane Proteins
3 types based on association with membrane:
1.Peripheral
2.Integral
3.Lipid-anchored
1. Peripheral membrane proteins
• weakly associated with membrane surface
• bind to polar lipid heads and/or to integral membrane proteins
–electrostatic interactions (ionic bonds and/or hydrogen bonds)
–easily extractable from membranes by high salt concentrations
(disrupting electrostatic interactions), or by EDTA (chelates Ca2+
and Mg2+)
• usually water-soluble
• Can also be fibrous proteins attached to membrane surface
(cytoskeletal proteins).
2. Integral membrane proteins
• tightly bound to membrane - interact with interior (membrane core, lipid
tails) (hydrophobic interactions)
• require detergents (or organic solvents) for extraction
• water-insoluble
• both peripheral and integral domains.
• can completely span membrane ("transmembrane proteins").
• Glycoproteins always have carbohydrates on extracellular side
side.
– carbohydrates attached by O-glycosidic
bonds to OH groups of Ser or Thr or
– N-glycosidic
bonds to Asn
Nelson & Cox, Lehninger
Principles of Biochemistry,
4th ed., Fig. 11-3
Membrane Proteins
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BIOC 460, Summer 2010
3.Lipid-anchored membrane proteins
• Covalently linked to lipids, required for association with membrane
• Can be reversibly attached to/detached from proteins.
– "switching device" to alter affinity of protein for membrane
– e.g., N-myristoylation: C14 fatty acid in amide
linkage to N
N-terminal
terminal Gly
examples:
–PKA (cAMPdependent
protein
kinase)
−α subunit of
G proteins
Nelson & Cox, Lehninger
Principles of Biochemistry,
4th ed., Fig. 11-3
Transmembrane Proteins
Hydrogen Bonding: an extremely important consideration for
inserting the polar backbone of a protein into a lipid membrane!
• By hydrogen-bonding all polar backbone groups via formation of
secondary structures, α-helical or β barrel motifs for membranespanning portions of transmembrane protein
protein, unfavorable
thermodynamic conditions are minimized.
Examples of Transmembrane Proteins demonstrating
transmembrane secondary structural motifs:
1. glycophorin A (erythrocyte membranes)
2. bacteriorhodopsin (purple membrane of Halobacterium halobium, a saltloving bacterium)
3. prostaglandin H2 synthase (COX, enzyme involved in biosynthesis of
prostaglandins/inflammatory response)
4. porins (channel-forming proteins -- outer membranes of gram-negative
bacteria and outer membranes of mitochondria and chloroplasts)
Membrane Proteins
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BIOC 460, Summer 2010
1. Glycophorin A:
• A glycoprotein: by mass ~60% carbohydrate, ~40% protein.
• Single transmembrane α-helix
• Most of protein (N-terminal portion) on outside of cell, exposed to water;
mainly hydrophilic residues, heavily glycosylated (carbohydrates in
glycosidic bonds to Ser, Thr, and Asn)
• Carbohydrates: ABO and MN blood group antigen-determining
structures.
• Extracellular part of protein also receptor for influenza virus binding to cells
• C-terminal portion on cytosolic side of membrane, interacts with
cytoskeletal proteins
19-AA
AA residue hydrophobic segment is exactly right length to span
• One 19
membrane if it’s coiled into an α-helix -- hydrophobic R groups
oriented outward, toward "solvent" (hydrophobic core of lipid bilayer).
• Hydrophobic 19-20 amino acid α-helices are very common way for
proteins to span biological membranes.
• Polar peptide backbone groups (carbonyl oxygens and amide N-H groups)
fully hydrogen-bonded
– Hydrogen-bonding "neutralizes" these polar groups, and
– "screens" them from contact with lipid core by R groups on outside of
helix.
30 Å across,
across so 20-residue
20 residue α
α-helix
helix (20
• hydrophobic core of membrane is ~30
residues x 1.5 Å "rise" per residue) is right length to reach across
hydrophobic core.
Berg et al., Fig. 12-27a
Membrane Proteins
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BIOC 460, Summer 2010
2. Bacteriorhodopsin
• from purple membranes of bacteria of the genus Halobacterium, salt-loving
archaebacteria
• 7 transmembrane α-helices (common motif for signaling proteins: "7-TM
helix receptors"
• 247 amino acid residues (26,000 MW) with retinal cofactor (absorbs light)
• uses light energy to pump protons across membrane, out of cell, against a
concentration gradient (example of primary active transport)
• generates and maintains [H+] gradient (pH gradient) across cell membrane
• transmembrane proton gradient = "stored" potential energy, used by
ATP synthase to drive ATP synthesis
Berg et al., Fig. 12-18
•globular shape,
most of protein
embedded in
membrane
b
•retinal (not shown)
in middle of bundle
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/rhodopsin/rhodop1.htm
2. Bacteriorhodopsin
• 7 TM helices closely packed; at 10o angle to plane of membrane
• outer sides of helices (interact with hydrophobic interior of membrane):
many hydrophobic R groups
• "inner" sides of helices have some charged/polar residues to interact
with retinal and for proton translocation
• Opposite of water-soluble globular proteins: in an integral membrane
protein not only are most R groups on interior of protein hydrophobic
protein,
hydrophobic, but
R groups on outside of protein are hydrophobic as well
Berg et al., Fig. 12-18
Membrane Proteins
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BIOC 460, Summer 2010
3. Prostaglandin H2 synthase
• COX, enzyme involved in biosynthesis of prostaglandins/inflammatory
response
• inhibited by aspirin and other NSAIDs
• integral membrane protein, homodimeric, with subunit structures primarily
α-helical
• does NOT span membrane -- it's on surface of endoplasmic reticulum (ER)
membrane extending into lumen of ER
membrane,
• firmly anchored in membrane by α-helices with hydrophobic R groups
Berg et al., Fig. 12-23
Berg et al.,
Fig. 12-24
3. PGH2 synthase
• Localization important to binding substrate, arachidonic acid, released from
membrane lipids by PLA2.
• Substrate can enter enzyme active site via hydrophobic channel (faces
membrane) without entering aqueous solvent.
• NSAIDs block channel, inhibit enzyme, prevent prostaglandin synthesis, and
reduce inflammation. (What type of inhibitor would naproxen be -competitive,
titi
noncompetitive,
titi
or uncompetitive?
titi ? What
Wh t is
i the
th
mechanism of Aspirins inhibitions of COX?)
Berg et al., Fig. 12-23
Membrane Proteins
Berg et al.,
Fig. 12-24
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BIOC 460, Summer 2010
4. Porins: a β−barrel for H2O transport
• channel-forming proteins in outer
membranes of gram-negative bacteria,
mitochondria, and chloroplasts
• antiparallel β−barrel, with hydrophobic
R groups facing membrane
R-groups
membrane, polar R
Rgroups facing interior of barrel,
stabilizing H2O.
What is significance of β-sheet with respect
to peptide backbone carbonyl O and
amide N-H groups?
Berg et al
al., Fig
Fig. 12
12-20
20
Jmol routine:
http://www.biochem.arizona.edu/classes/bioc462/462a
/jmol/porin/newporin.html
Berg et al., Fig. 2-50
Lipid Rafts or Microdomains
• In late 1990’s a modification of
the Fluid-Mosaic Model emerged
• On long timescales (msec – sec)
lipids diffuse freely
• On shorter times (µsec) lipids
appear
pp
localize,, then “hop”
p to
another region
• Phosphosphingolipids and
cholesterol seem to cluster
together in “rafts”
• ~50 nm diameter; significantly
thicker
• Few thousand lipids; 10 – 50
proteins
• Specificity
S
ifi it ffor ttype off proteins
t i
localized
• Organize receptors or signaling
proteins(?)
Membrane Proteins
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BIOC 460, Summer 2010
Rafting in the Sea of Lipid
Rafts can be visualized by Atomic
Force Microscopy (left). The
cantilever acts like the stylus arm on
an “ancient” record player.
Th peaks
The
k b
below
l
are GPI
GPI-linked
li k d
proteins protruding above raft
(green). Rest of membrane (black).
Learning Objectives
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Membrane Proteins
Terminology (as applied to membrane proteins): peripheral, integral,
lipid-anchored (operational definition -- how can they be extracted
from membrane?); trans-membrane helix; antiparallel β barrel
Briefly explain what “lipid rafts” are in membranes. What are primary
lipid components?
Explain in structural terms how an integral membrane protein can deal
with its polar backbone groups in spanning the hydrophobic core of a
lipid bilayer.
Name 2 types of secondary structural elements used by integral
membrane proteins to cross membranes. Describe where the R
groups are located in these secondary structural elements relative to
the hydrophobic lipid core.
Discuss the structural properties of the following examples of
membrane proteins: glycophorin A, bacteriorhodopsin, prostaglandin
H2 synthase, and a porin. Include in your discussion the type(s) of
secondary structure and types of R groups found in the
transmembrane (membrane-spanning) structural components of these
membrane proteins.
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