lecture 16: membrane proteins. membrane proteins: 1) overview: types and properties 2) getting into...
TRANSCRIPT
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Lecture 16: Membrane Proteins
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Membrane proteins:1) Overview: Types and Properties
2) Getting into the Membrane3)What Membrane Proteins Do--
examples3) Working with Membrane Proteins
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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
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Beta-Barrel
Alpha Helix
Amphipathic Helix
Fatty acid Chain
GPI-Anchored
Peripheral
Types of Membrane Proteins
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Beta-Barrel Proteins
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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.
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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.
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Hydropathy Plots: New Method
Von Heijne, Biochem Soc. Trans. 2011
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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.
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Membrane proteins:1) Overview: Types and Properties
2) Getting into the Membrane3)What Membrane Proteins Do--
examples3) Working with Membrane Proteins
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Getting into the Membrane: Destination Matters!
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Challenges of co-translational protein targeting
Lots of ribosomes Select the right ribosomeTranslation is fast
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SRP and its receptor act as molecular matchmakers
SR
SRP
SR
ribosome
SRP
ribosome
SRP
SRribosome
SRPribosome
7-12 residue hydrophobic
corebasic
N-terminus
Signal peptides
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The core protein targeting machineryis conserved
Eukaryotic (Human)
Bacterial (E. coli)Ffh FtsY
SR
SR
SRP54
SRP68
SRP72SRP19
SRP9
SRP14
SRP SRP Receptor
SRP-Type GTPases
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QuickTime™ and ampeg4 decompressor
are needed to see this picture.
Crystal Structure of the Prokaryotic SRP/SR
Ataide et al., Science 2011
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Mg
a.w.
Mg
GMPPCP
GMPPCP
a.w.
D139
D135
SRP
SR
Egea et. al.,Nature, 2004 and Focia et al., Science, 2004
SRP and SR are GTPases
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GTP hydrolysis and the SRP targeting cycle
SR
SRP
SR
ribosome
ribosome
SRP
SRP
SRribosome
GTP
GTP GTPGTP
GDP
GDP
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Co-translational protein targeting: Structure of RNC-translocon complex
Frauenfeld et al., PNAS 2011
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Structure of the Translocon
van den Berg et al., Nature 2004
Top view Side view
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Translocon features: the plug and the lateral gate
van den Berg et al., Nature 2004
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Translocon activity!
van den Berg et al., Nature 2004
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Getting into the ER: Single N-terminal TM Segment
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Getting into the ER: Positive Inside Rule
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The Problem with Tail Anchored Proteins:
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Post Translational Targeting: Tail Anchored Proteins
Hedge and Keenan, 2011
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The GET Complex Targets Tail-Anchored Proteins
Hedge and Keenan, 2011
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Post-translational Protein Targeting in Prokaryotes
Cross et al., 2009
Very hydrophobic and basic signal peptides go through SRP-dependent pathway. Others go through SecB
Folded proteins go through the Tat pathway
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Model for SecA-mediated Protein Translocation
Zimmer et al., Nature 2008
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.
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Twin Arginine Transporter: Transports Folded Proteins
Palmer and Berks Nature Reviews Microbiology 2012
Signal sequence contains twin arginines
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Chloroplasts and Mitochondria Have Their Own Genome but Most Proteins are Made in the Cytosol and Imported.
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Trafficking to Mitochondrial Membranes
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Membrane proteins:1) Overview: Types and Properties
2) Getting into the Membrane3)What Membrane Proteins Do--
examples4) Working with Membrane Proteins
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Functions of Membrane Proteins:
SignalingTransportersEnzymesAnchors
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Signaling: IRE1 and the Unfolded Protein Response
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Signaling: IRE1 and the Unfolded Protein Response
Kawaguchi and NG, Science 30 September 2011
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Transporters: Aquaporins
Gonen et al., Nature 2005
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Enzymes: FtsH, a Membrane Bound Protease
Krzywda et al., Structure 2002 and Bieniossek et al., PNAS 2009
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Membrane proteins:1) Overview: Types and Properties
2) Getting into the Membrane3)What Membrane Proteins Do--
examples4) Working with Membrane Proteins
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Working with Membrane Proteins: Pick Detergents Wisely
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Proteoliposomes
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Hegde RS, Keenan RJ Nature Reviews cell Biology 2011.
Not Many Membrane Protein Crystal Structures Solved
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Lipidic Cubic Phase for Membrane Proteins
Ehud M. Landau and Jürg P. Rosenbusch, PNAS 1996, and Nollert et al., FEBS Letters 2001
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Electron Crystallography: An Example (Wza)
Nesper et al.,JBC 2003
Neg. Stain Diffraction Pattern
2-D crystals Class averages of single particles
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Electron Crystallography: Aquaporin
Gonen et al., Nature 2005
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Electron Crystallography: Solved Structures
Wisedchaisri et al., Nature 2005