V6 Membrane Beta Barrels – Membrane Positioning

V6 SS 2009Membrane Bioinformatics

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V6 Membrane Beta Barrels – Membrane Positioning

Beta-barrels are the second important type of

transmembrane proteins.

They are mostly found in the outer membranes

of bacteria, chloroplasts, and mitochondria.

They function as:

(1) Simple passive pores for transport across

bacterial membranes

(2) active ion transporters for nutrient uptake,

membrane anchors, defense against

pathogenic proteins.

Schulz, Curr Opin Struct Biol 10, 443 (2000)

Georg Schulz (Uni Freiburg):First X-ray structure of porin (1992)


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(1) Schulz: 10 roles for Membrane Beta Barrels1. The number of β-strands is even. The N and C terminiare at the periplasmic barrel end.

2. The β-strand tilt is always around 45° and correspondsto the common β-sheet twist. Only one of the two possibletilt directions is assumed, the other one is an energeticallydisfavored mirror image. (Today: tilt between 20 and 45°)

3. The shear number of an n-stranded barrel is positiveand around n+2, in agreement with the observed tilt.

4. All β-strands are antiparallel and connected locally to their next neighbors along the chain, resulting in a maximum neighborhood correlation.

5. The strand connections at the periplasmic barrel end areshort turns of a couple of residues named T1, T2 and so on.



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Schulz: 10 roles for Membrane Beta Barrels6. At the external barrel end, the strand connections areusually long loops named L1, L2 and so on.

7. The β-barrel surface contacting the nonpolar membraneinterior consists of aliphatic sidechains forming a nonpolarribbon with a width of about 22 Å.

8. The aliphatic ribbon is lined by two girdles of aromaticsidechains, which have intermediate polarity and contactthe two nonpolar–polar interface layers of the membrane.

9. The sequence variability of all parts of the β-barrel duringevolution is high when compared with soluble proteins.

10. The external loops show exceptionally high sequencevariability and they are usually mobile.



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shear

Ideal topology, see Fig. on the right.

However, TM -strands do not span the membrane

at 90° (perpendicular to the membrane).

They are usually inclined at an angle to the vertical TM axis.

This results in a shift in the H-bonded residues, termed the shear number.

A shear number of +1 means that the H-bonded partner of the residue at position i

is at position j + 1 rather than j.

Waldispühl et al. Proteins 65, 61 (2006)


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Structures of Membrane Beta Barrels

Nowadays: β-barrels size from small 8-stranded to large 22-stranded proteins.

Oligomerization state: TMBs can against exist as monomers or oligomers.

Their topology is defined by the strand number and shear number (measure of

inclination angle of beta-strand against the axis).


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partiFold

Model is motivated by an abstract physical description of

omps.

It uses -strand contact energy parameters for globular

proteins taken from the program BETAWRAP

[statistical potentials: W(r) = -kT ln p(r)]

Jerome Waldispühl (MIT)


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Structural features

Fundamental features of beta-barrel structures:

(i) The overall shape of the barrel (# of strands, their relative arrangement)

(ii) A list of antiparallel -strand pairs; residue contacts and side chain orientation

(iii) Inclination of TM -strands through the membrane plane.



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2-tape representation

Decomposition of TMB into individual blocks of antiparallel -strands.

Each strand is involved in two „pairings“.

Figure shows 2-tape representation.

Pairings are made from one tape to the other.



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New notation

Each block is represented as 4-tuple

22

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ji

ji

where i1 and j1 are the indices of the strand on the first tape and i2 and j2 are those

on the second tape.

M : -strand residues with side-chains oriented toward the membrane.

C : residues with side-chain oriented toward the channel.

E : unpaired -strand residues


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partiFold

Model is based on an abstract physical description of omps.

It uses -strand contact energy parameters for globular proteins taken from the

program BETAWRAP.



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partiFold: computation of structures

Compute energies of all conformations

using statistical potential for amino acid

stacking pairs.

Use dynamic programming approach to

sample all possible TMB structures,

compute their energies, and thus the

partition function.

partiFold algorithms then predicts an

ensemble of structural conformations for a

TMB.

Energy function apparently needs to be

refined further ...



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Another interesting approach: statistics of NP-patterns

Shown here:

Pattern frequencies in

Soluble proteins.

Need to perform

analogous statistics

for TM barrels.

(ongoing work

by Sikander)

Mandel-Gutfreund,

Gregoret,

JMB 323, 453 (2002)

Membrane Bioinformatics – Part II13

TMHMM: 1000 of 4288 predicted E.coli

genes are inner membrane proteins.

737 genes encode proteins with > 100

residues and 2 TM helices.

714 were suitable for cloning into phoA

and gfp fusion vectors.

Both fusions could be obtained for 573

genes, one fusion for an additional 92

genes.

(2) Global Topology Analysis

Daley et al. Science 308, 1321 (2005)

Knowing the topology of a TM protein is

essential to understanding its function.

Idea: generate reference point, e.g.

the location of a protein‘s C terminus.

E.coli attach alkaline phosphatase

(PhoA) to C-terminus that is active only

in the periplasm of E.coli, or green

fluorescent protein (GFP) that

fluoresces only in the cytoplasm.


Using homology, 601 proteins could be

assigned a topology.

For 71 of these, the location of the C terminus

was already established.

The results agreed except for 2 cases.

The error rate is therefore ~ 1%.

TMHMM alone predicts the correct C-terminal

location for 78% of the 601 proteins.

By providing unambiguous C-terminal

locations, the TMHMM reliability score

increases for 526 proteins and decreases for

75 proteins.

Global Topology Analysis



Functional categorization of E.coli inner membrane proteome


clear trend for Nin – Cin topologies (even number of TMH)

- largest functional category is transport proteins, many with

6 or 12 TM helices.

Most proteins with unknown function have 6 TM helices.


Idea: transfer experimental data set from PhoA and GFP-fusions of 608 proteins to

homologous proteins.

In March 2005 were available, 204 annotated eubacterial and 21 archeal genomes,

with 658,210 annotated protein sequences.

Perform BLAST searches (E-value < 10-5)

30,744 sequence hits where TMHMM predicts 1 TM helix

Second BLAST query with these 30,744 sequences

17,111 „secondary homologs“.

Extend predictions by sequence homology

Granseth et al., J.Mol.Biol. 352, 489 (2005)


Unconstrained vs. constrained prediction

Granseth et al., J.Mol.Biol. 352, 489 (2005)

(a) Unconstrained TMHMM predictions for the

full set of 158,182 sequences with 1 predicted

TM helix (grey bars) and constrained predictions

for the 51,208 sequences for which the C-

terminal location or the location of an internal

residue could be annotated (black bars).

The number of proteins with different topologies

are shown; Cin topologies are plotted upwards,

Cout downwards. The number of Cout proteins with

a single TM helix (39,322) is off-scale.

The unconstrained algorithm predicts too many

proteins as Cout.

(b) TMHMM predictions for the 51,208 annotated

sequences before (grey bars)

and after (black bars) constraining the

predictions with the location of the annotated

residue.


Most TM proteins are expected to adopt only one topology in the membrane.

Global topology analysis of E.coli inner membrane proteome identified 5 dual-

topology candidates: EmrE, SugE, CrcB, YdgC, YnfY.

All are quite small (~ 100 aa), contain 4 strongly predicted TM segments, contain

only few K and R residues and have very small (K + R) bias.

(3) Dual-topology proteins?

Rapp et al., Nat.Struct.Biol. 13, 112 (2006)

(a) A dual-topology protein inserts into the membrane in two opposite directions. As nearly all helix-bundle membrane proteins have a higher number of lysine (K) and arginine (R) residues in cytoplasmic (in) than in periplasmic (out) loops (the ‚positive-inside‘ rule), dual-topology proteins are expected to have very small (K + R) biases.

Rectangles: TM segmentsblack dots: K and R residues


Without solving their 3D structures, how can one prove that a protein has dual

topology?

Such a protein would be particularly sensitive to the addition or removal of a single

positively charged residue in a loop or tail.

measure activities of two different, C-terminally fused reporter proteins:

PhoA (only enzymatically active when in the periplasm)

GFP (fluorescent only when in the cytoplasm).

Concentrate on N-terminus and first loop.

Dual-topology proteins?



(a) wt YdgE-PhoA fusion is active,

wt YdgE-GFP fusion is inactive

C-terminus in periplasm (Cout )

wt YdgF behaves oppositely (Cin)

These 2 proteins are topologically

stable.

(b – d) C-terminal orientation of

EmrE, SugE, CrcB, YnfA and

YdgC is highly sensitve to charge

mutations.

For 14 or 19 charge mutations,

both PhoA and GFP activities

change in the direction expected

from the change in (K + R) bias.

Charge mutations shift the orientations of dual-topology TM proteins



Experimental techniques to study orientation of proteins in membranes are:

- chemical modification

- spin-labeling

- fluorescence quenching

- X-ray scattering

- neutron diffraction

- electron cryomicroscopy

- NMR

- polarized infrared spectroscopy.

It is very desirable to complement them by computational methods.

- e.g. explicit-solvent molecular dynamics simulations

- here: simplified approach that minimize the protein transfer energy

from water to a hydrophobic slab used as a membrane model.

(4) Positioning of proteins in membranes – OPM database

Adamian & Liang, Proteins 63, 1 (2006)


important parameters

Lomize et al. Prot.Sci. 15, 1318 (2006)


Model protein as a rigid body that freely floats in the planar hydrocarbon core of

a lipid bilayer.

Calculation of transfer energy


ii

MW

iitransferzfASAdzG ,,,

0

ASAi : accessible surface area of atom i

iW-M : solvation parameter of atom i (transfer energy of the atom from water to

membrane interior in kcal/(mol.Å2) )

f(zi): interfacial water concentration profile with = 0.9 Å

0

1

1zzi i

ezf


ionization of charged residues

Residues that are typically charged in soluble proteins may become neutral in the

hydrophobic inside of the bilayer!

The ionization/protonation energies of charged residues are described by the

Henderson-Hasselbalch equation:


aioniz

pKpHRTG 3.2at pH = 7

average pKa value Gioniz

in proteins [kcal/mol]

Arg 12.0 6.9

Lys 10.4 4.7

Asp 3.4 4.9

Glu 4.1 4.0

His 6.6 0.6


use deterministic 2-step search strategy:

(1) grid scan to determine a set of low-energy combinations of variables z0, d, , grid steps: 0.5 Å for z0 and d, 5° for , 2° for

(2) local energy minimization (Davidon-Fletcher-Powell method) starting from low-

energy points

Also consider energetically best rotation of solvent-exposed charged side chains

(e.g. Lys and Arg) that are situated close to the calculated boundaries and could

be rotated away from the hydrophobic core snorkeling.

Global energy optimization



Which solvation parameters to use?

chx and dcd results agree well with experiment, oct agrees poorly.



Pay attention to …

slightly different parameter sets should be applied for proteins in detergents and

bilayers

Gtransfer should not include contributions of atoms that face internal polar cavities

of TM proteins and that do not directly interact with surrounding bulk lipid

Otherwise, the orientation of many -barrels and pore-forming transporters would

be computed incorrectly



Main features of model

necessary and sufficient approximations for reproducing the exp. Data

(1) lipid bilayer is represented as planar hydrophobic slab with adjustable

thickness and a narrow interfacial area with a sigmoidal polarity profile

(2) proteins are considered as rigid bodies with flexible side chains; their transfer

energies are minimized with respect to 4 variables

(3) transfer free energy is calculated at an all-atom level using atomic solvation

parameters determined for the water-decadiene system

(4) neglect explicit electrostatic interactions, account for neutralization of charged

residues

(5) eliminate contributions of pore-facing atoms



parameters of model

The model only depends on 5 atomic solvation parameters (N, O, S, sp2 C,

sp3 C), one constant , and the ionization energies of charged groups.

All can be obtained independently from experimental sources.

Verify method for 24 TM proteins of known 3D structure whose spatial position in

bilayers have been exp studied.



Average tilt angles

(a) hydrophobic thickness matches well (table 2)


(b) the calculated tilt values are in excellent agreement with NMR data,

they also correlate well with ATR-FTIR data (table 3), although the exp. values are

systematically larger orientational disorder in the experiments?


Membrane penetration depths



Biological membranes differ



Membrane pentration depths



Membrane core boundaries


Additional slides


application to all other 109 TM protein complexes

80 -helical

28 -barrels

gramicidin dimer

control set:

20 water-soluble proteins

32 monotopic and peripheral proteins

Application to all TM proteins from the PDB



Peripheral and monotopic

proteins have low penetration

depths.

Calculated tilt angles vary

from 0° - 6°.

TM proteins tend to be

nearly perpendicular to the

membrane, although the

individual helices are on

average tilted by 21°.

Application to membrane proteins



Global topology analysis of E.coli inner membrane proteome showed that ca. 20 –

25% of the TM proteins have 10 TM helices.

These are often involved in transport of small molecules across the membrane.

Many of these proteins will have buried helices. Can we identify those?

Develop an empirical helix burial function f based on a few assumptions.

(i) residues in buried helices are more conserved because of structural and

functional contraints.

(ii) the residue composition of the buried helices is different from the composition of

helices facing the lipid environment.

(iii) the difference between the minimal and maximal values of conservation

entropy for every position in MSAs of TM helices should be smaller in buried

helices than in lipid-exposed helices because of the homogenous environment.

(4) Prediction of buried TM helices



f: burial function

s: average entropy of all residue positions of the TM helix

l : average lipophilicity

k: sorted entropy values of all residue positions in a helix of length d for helices

1 ... n of the TM protein

Problems: the average entropy depends on the number of sequences in the MSA.

needs MSAs with exactly the same set of sequences from the same set of

species.

Also, the stability of different membrane proteins in the lipid environment may be

different.

Account for ambiguity in the definition of TM helix ends.

Burial Function


lskf

d

ssss d

...21

d

llll d

...21


Ranking of TM helices by burial function and robustness



(a) TM helices TM4, TM5, TM6, TM8 form core, consistent with prediction.

(b) TM4, TM10 are most buried.

(c) one can explain prediction of TM8 as buried by considering a tightly bound

cardiolipin molecule identified in the X-ray structure.

Examples of buried TM helices that are correctly predicted



Is the method applicable to TM

proteins where only sequence data

is available?

Test on structure of Leu transporter.

TMHMM predicts 12 TM helices.

Good overlap with X-ray helices.

Problem that no additional

sequences exist that are annotated

as Na+-dependent Leu transporters.

LeuTAa has 3 significantly buried

helices: 1, 6 and 8.

1 and 6 are true positives, 2 is a

false positive, 8 is a false negative.

Test ranking results



Pfam searches in 174 fully sequenced bacterial genomes for homologs (E < 10-10)

to SugE, EmrE, YdgE, CrcB, YnfA, YdgC and YdgO/YdgL.

Create multiple sequence alignment with ClustalW.

Use TMHMM to predict the positions of TM helices.

Obtain consensus TM helix prediction, compute (K + R) biases for individual

proteins. 10 residues from each of the flanking TM helices were included to allow

for possible misprediction of the exact positions of the loop ends.

Dual-topology homologs occur as gene pairs or singletons



Interpretation: SMR and CrcB occur as closely spaced pairs or as singletons.

Paired genes encode homologous proteins with opposite (K + R) bias.

Dual-topology homologs occur as gene pairs or singletons



Most likely evolutionary scenario:

a single dual-topology protein

undergoes gene duplication, the

two resulting proteins become

fixed in opposite orientations and

finally fuse into a single

polypeptide.

An internally duplicated protein with opposite topology


V6 Membrane Beta Barrels – Membrane Positioning

Documents

membrane anchors

membrane bioinformaticsv6

membrane beta barrels1

membrane beta barrels6

strand number

stranded proteins

strand tilt

strand connections