Lecture 16: Membrane Proteins

Membrane proteins:1) Overview: Types and Properties

2) Getting into the Membrane3)What Membrane Proteins Do--

examples3) 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

Beta-Barrel

Alpha Helix

Amphipathic Helix

Fatty acid Chain

GPI-Anchored

Peripheral

Types of Membrane Proteins

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 Membrane3)What Membrane Proteins Do--

examples3) Working with Membrane Proteins

Getting into the Membrane: Destination Matters!

Challenges of co-translational protein targeting

Lots of ribosomes Select the right ribosomeTranslation is fast

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

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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Crystal Structure of the Prokaryotic SRP/SR

Ataide et al., Science 2011

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

GTP hydrolysis and the SRP targeting cycle

SR

SRP

SR

ribosome

ribosome

SRP

SRP

SRribosome

GTP

GTP GTPGTP

GDP

GDP

Co-translational protein targeting: Structure of RNC-translocon complex

Frauenfeld et al., PNAS 2011

Structure of the Translocon

van den Berg et al., Nature 2004

Top view Side view

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 N-terminal 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 Tail-Anchored Proteins

Hedge and Keenan, 2011

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

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.

Twin Arginine Transporter: Transports Folded Proteins

Palmer and Berks Nature Reviews Microbiology 2012

Signal sequence contains twin arginines

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 Membrane3)What Membrane Proteins Do--

examples4) Working with Membrane Proteins

Functions of Membrane Proteins:

SignalingTransportersEnzymesAnchors

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 Membrane3)What Membrane Proteins Do--

examples4) Working with Membrane Proteins

Working with Membrane Proteins: Pick Detergents Wisely

Proteoliposomes

Hegde RS, Keenan RJ Nature Reviews cell Biology 2011.

Not Many Membrane Protein Crystal Structures Solved

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)

Nesper et al.,JBC 2003

Neg. Stain Diffraction Pattern

2-D crystals Class averages of single particles

Electron Crystallography: Aquaporin

Gonen et al., Nature 2005

Electron Crystallography: Solved Structures

Wisedchaisri et al., Nature 2005