Membrane Bioinformatics SoSe 2009 Böckmann & Helms

V1 SS 2009

Membrane Bioinformatics1

Membrane Bioinformatics SoSe 2009

Böckmann & Helms

V1 SS 2009


What is “Membrane Bioinformatics” ?

Increasing interest in structure & function of membrane proteins (ion

channels, G-protein coupled receptors), but only few structures are known

structure prediction of membrane proteins, prediction of function from

sequence

Function of Membrane Proteins: depends on membrane composition,

lipid-protein interactions, lipid mediated protein-protein interactions ...

Drug Transport through Membranes: depends on physico-chemical

membrane properties

Membranes may also play a direct role in signal transduction

Diseases associated with changes in lipid composition (diabetes,

schizophrenia, Tay-Sachs syndrome)

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Pharmaceutical relevance

Membrane proteins are crucial for survival:

- they are key components for cell-cell signaling

- they mediate the transport of ions and solutes across the membrane

- they are crucial for recognition of self.

The pharmaceutical industry preferably targets membrane-bound receptors.

Particularly important: large superfamily of G protein-coupled receptors (GPCRs)

- receptors for hormones, neurotransmitters, growth factors, light and

odor-related ligands.

More than 50% of the prescription drugs act on GPCRs.

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Lecture Content

Properties of Lipid Membranes (Rainer Böckmann)

Properties of Membrane Proteins (Volkhard Helms)

- Insertion of TM proteins into membrane: Translocon, MINS (today, V1)- Prediction of TM segments from sequence (V2)

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- Predicting lipid-facing helix faces from sequence: TMX (V5)- Predicting helix interactions from sequence (V6)

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- Classification of membrane protein function from sequence (V9)- Predicting the topology of beta-barrel proteins (V10)

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Physico-Chemical Properties of Membranes (Composition, Chemical Structure, Self-Organisation,

Phase Transitions)

S.J. Marrink and A.E. Mark JACS 125 (2003) 15233-15242

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Molecular Theory of Membranes (Chain Packing, Elasticity)

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Electrostatic Properties of Membranes and Ion Channels

R.A. Böckmann, A. Hac, T. Heimburg, H. Grubmüller Biophys.J. 85 (2003) 1647-1655

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Electroporation of Membranes

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Membrane-Protein Interactions, Role of Lipids

S.W.I.Siu and R.A. Böckmann (2009)

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Certification

Grade of certification (Schein) is based on an individual final oral exam.

Condition for the participation in the final oral exam:

(a) more than 50% of points from 4 assignments

(b) every student needs to present once in tutorial.

Assignments are given out after lectures

V2 (Helms), V4 (Böckmann), V6 (Helms), V8 (Böckmann).

Each assignment is to be completed within two weeks.

Up to two students can submit a solution.

Tutorial (every 2 weeks) will take place after submission of each assignment;

date to be decided.

Credit points: 5 (2V + 1Ü) for a special lecture in bioinformatics

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Role of the Membrane

Membranes enable formation of compartments!

Intracellular space is sub-divided (organelles, cytosol)

Distribution of different molecules among the subspaces

Membranes allow gradient of composition between nucleus and plasma membrane:

directed flow of newly synthesized material from ER to plasma membrane, trafficking of nutrition molecules in opposite direction

Membranes allow ionic/pH gradients in organelles: electrochemical gradient, activity control of specialized proteins (lysosomes), accumulation of specific

proteins O.G. Mouritsen Life – as a Matter of Fat Springer (2005)

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Architecture of the Plasma-Membrane

Plasma membrane

has 3 layers:

1. glycocalix: film formed by

oligosaccharides of

glycolipid head groups

2. center: lipid/protein layer

3. Intracellular side:

cytoskeleton

In this lecture, we will focus

on region 2.Addison-Wesley 1999

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Inside the hydrophobic core of the lipid bilayer, the protein backbone may not form

hydrogen bonds with the aliphatic chains of the phospholipid molecules

the backbone atoms need to form H-bonds among eachother.

they must adopt either -helical or -sheet conformations.

Topology of Membrane Proteins

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Lipid bilayer simplifies the prediction problem

TM proteins are forced into two classes: -helical, or -sheet.

-helices are typically tilted with respect to the membrane normal

between 10 – 45°.

The hydrophobic lipid bilayer reduces the three-dimensional structure formation

almost to a 2D problem.

http://www.biologie.uni-konstanz.de/folding/Structure%20gallery%201.html

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History of membrane protein structure determination1984 bacterial reaction center (Martinsried) noble price to Michel, Deisenhöfer, Huber 1987

1990 EM map of bacteriorhodopsin Henderson

1997 high-resolution structure by Lücke

now many intermediates of the photocycle

1992 porin (complete -barrel) Schulz (Freiburg)

1995 Cytochrome c Oxidase Michel (Frankfurt)

1998 F1ATPase noble price to John Walker 1997

1998 KCSA ion channel noble price to Roderick McKinnon 2003

2000 aquaporin

2000 rhodopsin (Palczewski)

2002 SERCA Ca2+ ATPase (Toyoshima)

2003 voltage-gated ion channel (McKinnon)

2008 First GPCR: adrenergic receptor

2009 P-glycoprotein (Chang)

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Partitioning across membranes

Partitioning from neutron diffraction data or from MD simulations.

White, von Heijne, Annu Rev Biophys (2008)

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Kyte-Doolittle hydrophobicity scale (1982)

Assign hydropathy value to each amino acid.

Use sliding-window to identify membrane

regions.

Sum the hydrophobicity scale over all

w residues in the window of length w.

Use threshold T to assign segment

as predicted membrane helix.

w = 19 residues could best discriminate

between membrane and globular proteins.

Threshold T > 1.6 was suggested for the

average over 19 residues.

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More refined indices

One drawback of pure hydropathy-based methods is that they fail to discriminate

accurately between membrane regions and highly hydrophobic globular segments.

-Wimley & White scale :

based on partition experiments of peptides

between water/lipid bilayer and water/octanol

http://blanco.biomol.uci.edu/hydrophobicity_scales.html

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Translocon-assisted insertion of TM proteins from Ribosome into ER membrane


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Crystal structure of translocon Sec YEG


Tom Rapoport(Harvard University)

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Integration of H-segments into the microsomal membrane

Hessa et al., Nature 433, 377 (2005)

b, Membrane integration of H-segments with the

Leu/Ala composition 2L/17A, 3L/16A and 4L/15A.

Bands of unglycosylated protein are indicated by a

white dot; singly and doubly glycosylated proteins are

indicated by one and two black dots, respectively.

Ingenious experiment! Introduce marker that shows whether helix segment H

is inserted into membrane or not.

a, Wild-type Lep has two N-terminal TM segments (TM1 and TM2) and a

large luminal domain (P2). H-segments were inserted between residues 226

and 253 in the P2-domain. Glycosylation acceptor sites (G1 and G2) were

placed in positions 96–98 and 258–260, flanking the H-segment. For H-

segments that integrate into the membrane, only the G1 site is glycosylated

(left), whereas both the G1 and G2 sites are glycosylated for H-segments

that do not integrate in the membrane (right).

Gunnar von Heijne(Stockholm University)

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Insertion determined by simple physical chemistry

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c, Gapp values for H-segments with 2–4 Leu residues.

Individual points for a given n show Gapp values obtained when the position of Leu is changed.

d, Mean probability of insertion (p) for H-segments with n = 0–7 Leu residues.

measure fraction of singly glycosylated (f1g) vs. doubly glycosylated (f2g) Lep molecules

appapp KRTG ln

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Biological and biophysical Gaa scales


a, Gappaa scale derived from H-segments with the indicated amino acid placed in

the middle of the 19-residue hydrophobic stretch.

Only Ile, Leu, Phe, Val really favor membrane insertion. All polar and charged

ones are very unfavored.

b, Correlation between Gappaa values measured in vivo and in vitro.

c, Correlation between the Gappaa and the Wimley–White water/octanol free

energy scale for partitioning of peptides.

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Positional dependencies in Gapp


a, Symmetrical H-segment scans with pairs of Leu (red), Phe (green), Trp (pink) or Tyr (light blue)

residues. The Leu scan is based on symmetrical 3L/16A H-segments with a Leu-Leu separation of one

residue (sequence shown at the top; the two red Leu residues are moved symmetrically outwards) up to

a separation of 17 residues. For the Phe scan, the composition of the central 19-residues of the H-

segments is 2F/1L/16A, for the Trp scan it is 2W/2L/15A, and for the Tyr scan it is 2Y/3L/14A. The G

app value for the 4L/15A H-segment GGPGAAALAALAAAAALAALAAAGPGG is also shown (dark

blue).

b, Red lines show G app values for symmetrical scans of 2L/17A (triangles), 3L/16A (circles), and

4L/15A (squares) H-segments.

c, Same as b but for a symmetrical scan with pairs of Ser residues in H-segments with the composition

2S/4L/13A.

Tyr and Trp are favorable in interface region.

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Summary – TM helix insertion

1. MPs are in thermodynamic equilibrium with the cell membrane’s lipid bilayer, which means that the stability and three-dimensional structure of MPs are ultimately determined by lipid-protein physical chemistry.

2. α-Helical MPs are identified during translation on the ribosome by the signal recognition particle that initiates docking of the ribosome to the membrane-embedded multi-protein translocon complex.

3. Elongating polypeptides from the ribosome pass through a translocon TM channel within the translocon complex.

4. The translocon’s U-shaped structure allows diversion of TM helices sideways into the lipid bilayer.

5. The diversion of the helices into the bilayer appears fundamentally to be a physicochemical partitioning process between translocon and bilayer.

6. The partitioning process can be described quantitatively by apparent free energies that serve as a code for the selection of TM helices by the translocon working in concert with the lipid bilayer.


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Summary – TM helix insertion

FUTURE ISSUES

1. Much more structural information about translocons and translocon

complexes is needed, especially an atomic-resolution structure of a

translocon engaged in polypeptide secretion.

2. Although there is a clear connection between the physical chemistry of

lipid-protein interactions and selection of TM helices by the translocon, a

quantitative molecular description of the empirical apparent free energies of

the translocon’s selection code is needed.

3. The molecular basis for the translocon-assisted assembly of multi-

spanning MPs needs to be established.


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Structure modelling for helical membrane proteins>P52202 RHO -- Rhodopsin. MNGTEGPDFYIPFSNKTGVVRSPFEYPQYYLAEPWKYSALAAYMFMLIILGFPINFLTLYVTVQHKKLRSPLNYILLNLAVADLFMVLGGFTTTLYTSMNGYFVFGVTGCYFEGFFATLGGEVALWCLVVLAIERYIVVCKPMSNFRFGENHAIMGVVFTWIMALTCAAPPLVGWSRYIPEGMQCSCGVDYYTLKPEVNNESFVIYMFVVHFAIPLAVIFFCYGRLVCTVKEAAAQQQESATTQKAEKEVTRMVIIMVVSFLICWVPYASVAFYIFSNQGSDFGPVFMTIPAFFAKSSAIYNPVIYIVMNKQFRNCMITT LCCGKNPLGDDETATGSKTETSSVSTSQVSPA

www.gpcr.org

EMBO Reports (2002)

1D

2D

3D

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MINS: predict membrane insertion G from sequence

Park & Helms, Bioinformatics 24, 1271 (2008)

Idea: amino acids in TM proteins accumulate at the most favorable regions

(1) Analyze distribution of amino acids at various membrane depth in all known

X-ray structures of TM proteins.

(2) Compute frequencies as a function of membrane depth

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MINS: membrane insertion G


To convert frequencies into free energies,

calibrate against exp. G for Hessa et al.

peptides.

r: frequency of amino acid i at depth z

ai(z) and bi: fit parameters for linear fit. Plot of MINS-predicted and experimentally measured membrane insertion free energies for 357 known cases

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MINS result for TM helices


TM helices and helices of secreted or cytoplasmic proteins are well separated!

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MINS1

Similar prediction as with MINS can be made with standard hydrophobicity scales:

WW: Wimely-White

KD: Kyte-Doolittle

GES:

EIS

But they give larger error than with MINS

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Performance of MINS1

to predict TM helices:

accuracycheck

TM helicesof polytopicTM proteins are not well predicted.

This indicatescooperativeinsertion ofTM helices

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Summary

TM proteins are a separate world; very different from soluble proteins.

Properties of TM proteins are intimately related to properties of the

surrounding lipid bilayer!

Structural Bioinformatics of Membrane Proteins is entering into a very

exciting phase right now.

Large interest of pharmaceutical companies due to recent availability of

new X-ray structures of adrenergic receptor, membrane transporters, ion

channels, and P-glycoprotein.

Structural data is now sufficient for developping data-driven bioinformatics

methods.

Documents

Membrane Bioinformatics SoSe 2009 Böckmann & Helms