Lecture 16: Membrane Proteins
Membrane proteins:
1) Overview: Types and Properties
2) Getting into the Membrane
3)What Membrane Proteins Do-examples
3) Working with Membrane Proteins
Membrane Protein Overview
-Membrane proteins are 25-35% of the
genome.
-Often important therapeutic targets:
involved in signaling, transport, etc.
-Can be anywhere from 25% (neurons) to
75% (mitochondria) by mass of the total
membrane.
-Under represented structurally
Types of Membrane Proteins
GPI-Anchored
Alpha Helix
Peripheral
Beta-Barrel
Fatty acid
Chain
Amphipathic Helix
Beta-Barrel Proteins
Alpha Helical Membrane Proteins
-Can classify by number of
transmembrane segments (multi
vs single pass) and their
topology in the membrane.
-Different methods are used to
predict if a polypeptide chain
will cross a membrane.
Hydropathy Plots for Alpha Helical
Proteins: Classic Method
For alpha-helical membrane
proteins, you can use
hydropathy plots to predict the
probability that a segment will
be within the membrane. These
are generated by measuring, for
each amino acid, its partition
coefficient between water and a
non-interacting, isotropic phase
such as ethanol, and calculating
from that partition coefficient a
transfer free energy.
Hydropathy Plots: New Method
Von Heijne, Biochem Soc. Trans. 2011
Membrane Attachment by a
Lipid Anchor
-Common for proteins involved in signaling.
-A fatty acid chain is attached via a amide or thioester
linkage, and anchors the protein in the membrane.
Membrane proteins:
1) Overview: Types and Properties
2) Getting into the Membrane
3)What Membrane Proteins Do-examples
3) Working with Membrane Proteins
Getting into the Membrane:
Destination Matters!
Challenges of co-translational protein
targeting
Select
Lots
Translation
the
of right
ribosomes
ribosome
is fast
SRP and its receptor act as molecular
matchmakers
Signal peptides
basic
N-terminus
ribosome
7-12 residue
hydrophobic
core
SRP
SRP
ribosome
SR
SRP
ribosome
SR
ribosome
SRP SR
The core protein targeting machinery
is conserved
SRP
SRP14
SRP9
SRP Receptor
SRP19
SRP72
SR
SR
SRP68
Eukaryotic
(Human)
SRP54
SRP-Type
GTPases
Ffh
FtsY
Bacterial
(E. coli)
Crystal Structure of the Prokaryotic
SRP/SR
QuickTime™ and a
mpeg4 decompressor
are needed to see this picture.
Ataide et al., Science 2011
SRP and SR are GTPases
SR
GMPPCP
D139
a.w.
Mg
Mg
D135
a.w.
SRP
GMPPCP
Egea et. al.,Nature, 2004 and Focia et al., Science, 2004
GTP hydrolysis and the SRP targeting cycle
ribosome
SRP
GTP
SRP
GDP
ribosome
SR
GTP
SRP
SR
GTPGTP
ribosome
SR
GDP
Co-translational protein targeting:
Structure of RNC-translocon complex
Frauenfeld et al., PNAS 2011
Structure of the Translocon
Top view
Side view
van den Berg et al., Nature 2004
Translocon features: the plug and the
lateral gate
van den Berg et al., Nature 2004
Translocon activity!
van den Berg et al., Nature 2004
Getting into the ER: Single Nterminal TM Segment
Getting into the ER: Positive Inside
Rule
The Problem with Tail Anchored Proteins:
Post Translational Targeting: Tail
Anchored Proteins
Hedge and Keenan, 2011
The GET Complex Targets TailAnchored Proteins
Hedge and Keenan, 2011
Post-translational Protein Targeting in
Prokaryotes
Very hydrophobic and basic
signal peptides go through
SRP-dependent pathway.
Others go through SecB
Folded proteins go through
the Tat pathway
Cross et al., 2009
Model for SecA-mediated Protein
Translocation
How does SecA use the energy of ATP hydrolysis to push a
polypeptide through the translocon? Structural studies
suggest that a “two helix finger” with a tyrosine paddle
pushes the polypeptide into the translocon. It’s actions are
coordinated with that of the peptide binding clamp, which
has different conformational states during ATP hydrolysis.
Zimmer et al., Nature 2008
Twin Arginine Transporter: Transports
Folded Proteins
Signal sequence contains twin
arginines
Palmer and Berks Nature Reviews Microbiology 2012
Chloroplasts and Mitochondria Have
Their Own Genome but Most Proteins
are Made in the Cytosol and Imported.
Trafficking to Mitochondrial
Membranes
Membrane proteins:
1) Overview: Types and Properties
2) Getting into the Membrane
3)What Membrane Proteins Do-examples
4) Working with Membrane Proteins
Functions of Membrane Proteins:
Signaling
Transporters
Enzymes
Anchors
Signaling: IRE1 and the Unfolded
Protein Response
Signaling: IRE1 and the Unfolded
Protein Response
Kawaguchi and NG, Science 30 September 2011
Transporters: Aquaporins
Gonen et al., Nature 2005
Enzymes: FtsH, a Membrane Bound
Protease
Krzywda et al., Structure 2002 and Bieniossek et al., PNAS 2009
Membrane proteins:
1) Overview: Types and Properties
2) Getting into the Membrane
3)What Membrane Proteins Do-examples
4) Working with Membrane Proteins
Working with Membrane Proteins: Pick
Detergents Wisely
Proteoliposomes
Not Many Membrane Protein
Crystal Structures Solved
Hegde RS, Keenan RJ Nature Reviews cell
Biology 2011.
Lipidic Cubic Phase for Membrane
Proteins
Ehud M. Landau and Jürg P. Rosenbusch, PNAS 1996, and Nollert et al.,
FEBS Letters 2001
Electron Crystallography: An Example
(Wza)
Neg. Stain
Diffraction Pattern
2-D crystals
Class averages of single particles
Nesper et al.,JBC 2003
Electron Crystallography: Aquaporin
Gonen et al., Nature 2005
Electron Crystallography: Solved
Structures
Wisedchaisri et al., Nature 2005