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V7 SS 2006
Membrane Bioinformatics – Part II1
V7 – Positioning of TM proteins in membrane
In the absence of high-resolution 3D structures, an important cornerstone for the
functional analysis of any membrane protein is an accurate topology model.
Topology model: describes the number of TM spans and the orientation of the
protein relative to the lipid bilayer.
Topology models can be generated by sequence-based prediction or by time-
consuming experimental approaches.
2 medium length assignments
tutorials Fri 13.00 - 14.30
V7 SS 2006
Membrane Bioinformatics – Part II2
Idea: generate reference point, e.g. the location of a protein‘s C terminus.
In E.coli attach alkaline phosphatase (PhoA) that is active only in the periplasm of
E.coli, or green fluorescent protein (GFP) that fluoresces only in the cytoplasm.
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.
(1) Global Topology Analysis
Daley et al. Science 308, 1321 (2005)
V7 SS 2006
Membrane Bioinformatics – Part II3
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
Daley et al. Science 308, 1321 (2005)
V7 SS 2006
Membrane Bioinformatics – Part II4
Functional categorization of E.coli inner membrane proteome
Daley et al. Science 308, 1321 (2005)
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.
V7 SS 2006
Membrane Bioinformatics – Part II5
Idea: transfer experimental data set from PhoA and GFP-fusions to homologous
proteins. Data on 608 proteins.
204 annotated eubacterial and 21 archeal genomes in March 2005,
658,210 sequences. 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)
V7 SS 2006
Membrane Bioinformatics – Part II6
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.
V7 SS 2006
Membrane Bioinformatics – Part II7
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.
(2) 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
V7 SS 2006
Membrane Bioinformatics – Part II8
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?
Rapp et al., Nat.Struct.Biol. 13, 112 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II9
(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
Rapp et al., Nat.Struct.Biol. 13, 112 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II10
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
Rapp et al., Nat.Struct.Biol. 13, 112 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II11
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
Rapp et al., Nat.Struct.Biol. 13, 112 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II12
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
Rapp et al., Nat.Struct.Biol. 13, 112 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II13
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.
(3) Prediction of buried TM helices
Adamian & Liang, Proteins 63, 1 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II14
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
Adamian & Liang, Proteins 63, 1 (2006)
lskf
d
ssss d
...21
d
llll d
...21
V7 SS 2006
Membrane Bioinformatics – Part II15
Ranking of TM helices by burial function and robustness
Adamian & Liang, Proteins 63, 1 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II16
(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
Adamian & Liang, Proteins 63, 1 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II17
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
Adamian & Liang, Proteins 63, 1 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II18
Experimental techniques to study orientation of proteins in membranes
chemical modification
spin-labeling
fluorescence quenching
X-ray scattering
neutron diffraction
electron cryomicroscopy
NMR
polarized infrared spectroscopy.
Desirable to complement by computational methods.
e.g. explicit-solvent molecular dynamics
... up to simplified approaches that minimize the protein transfer energy
from water to a hydrophobic slab (used as a membrane model).
(4) Positioning of proteins in membranes
Adamian & Liang, Proteins 63, 1 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II19
important parameters
Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II20
Model protein as a rigid body that freely floats in the planar hydrocarbon core of a
lipid bilayer.
Calculation of transfer energy
Adamian & Liang, Proteins 63, 1 (2006)
ii
MW
iitransferzfASAdzG ,,,
0
ASAi : accessible surface area of atom i, computed with NACCESS
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
V7 SS 2006
Membrane Bioinformatics – Part II21
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:
Lomize et al. Prot.Sci. 15, 1318 (2006)
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
V7 SS 2006
Membrane Bioinformatics – Part II22
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
Global energy optimization
Adamian & Liang, Proteins 63, 1 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II23
Which solvation parameters to use?
chx and dcd results agree well with experiment, oct agrees poorly.
Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II24
features of model
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
( mention results of Sam)
Otherwise, the orientation of many -barrels and pore-forming transporters would
be computed incorrectly
Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II25
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
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.Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II26
Average tilt angles
(a) hydrophobic thickness matches well (table 2)
Lomize et al. Prot.Sci. 15, 1318 (2006)
(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?
V7 SS 2006
Membrane Bioinformatics – Part II27
Membrane penetration depths
Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II28
Introduction
Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II29
Membrane core boundaries
Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II30
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
Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II31
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
Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II32
Biological membranes differ
Lomize et al. Prot.Sci. 15, 1318 (2006)
V7 SS 2006
Membrane Bioinformatics – Part II33
Fluctuations are larger for TM proteins with
a smaller TM perimeter.
Fluctuations around energy minimum
Lomize et al. Prot.Sci. 15, 1318 (2006)