Download Escherichia coli

Melissa David
Adam Ossin
Rutger Mantingh
Supervisor:
Antoinette Killian
Introduction
Integral membrane proteins
 in Escherichia coli cells
 Located in the inner membrane
of cell wall
 Vital for cellular functions
• Difficult to study
 Due to hydrophobic and
amphiphilic nature
 Less than 1% of high resolution
3D structures known
Periplasm
Inner
membrane
Cytoplasm
Which alternative method could be
used to study integral proteins?
Membrane topology prediction
Topology model


Membrane topology describes which regions
of a polypeptide spans the cell membrane
Membrane topology can be predicted


protein sequence
Membranes were thought to have only one
topology
How did they prove that dual topology
proteins may exist?
With the use of (K + R) biases as
determinant for membrane protein
topology
(K + R) bias determination



Orientation of membrane is
determined
Loops in cytoplasm has more
positive charged residues
(‘positive-inside rule’)
Effect of single positively charged
residue


(K+R) bias close to zero  change
in orientation of protein
Considerable (K+R) bias  no
effect on orientation of protein
Dual topology proteins



Dual topology membrane proteins
 Inserts into the membrane in 2
opposite orientation
Five candidates for dual-topology:
 EmrE, SugE, CrcB, YdgC, YnfA
features of these proteins
 Quite small ~ 100 amino acid
residues
 4 transmembrane helices
 Only few positively charged
lysine and arginine residues
 Very small (K + R) bias between
loops
Evolutionary relationship of membrane proteins
Singleton
Gene
pair
Protein 1 Protein 2
Protein 1 Protein 2
Gene
pair
Singleton
Protein 1 Protein 2
Protein 1 Protein 2
Hypothesis

Dual topology proteins have no or a very
small positive amino acid bias. Therefore,
adding or subtracting a single positive
amino acid will result in topology changes.
Methods(1) How to determine topology?

Fusion proteins on Cterminus:

PhoA
PhoA: enzymatically active
only in the preiplasm
GFP

PhoA
GFP: florescent only in
cytoplasm
GFP
Methods (2)

How to determine
biases?

Unbiased proteins
are incorporated
either way (dualtopology)

Biased proteins are
incorporated in one
way
Methods (3) Mutations
Addition or
substitution
of/with
positive amino
acids (K + R)
SugE and EmrE
EmrE &
SugE
SugE and EmrE
PhoA
EmrE &
SugE
GFP
PhoA
GFP
SugE and EmrE
PhoA
EmrE &
SugE
GFP
PhoA
GFP
Control
YdgE
YdgF
CrcB
CrcB
YnfA and YdgC
YnfA
YdgC
Dual topology proteins: A single gene or a gene pair
Singleton
Singleton
Protein
Protein
1 Protein
1 Protein
2
2
Gene
Gene
pairpair
Protein
Protein
1 Protein
1 Protein
2
2
225 fully sequenced genomes scanned for pairs and
singletons
Determination by :
SMR protein family
-Both singletons and gene pairs
-Singletons around (K+R) bias = 0
-Gene pairs bigger (K+R) bias
(Leucine + Arginine) bias of “dual topology” proteins
SMR protein
family
Singleton
Protein 1 Protein 2
Gene pair
Protein 1 Protein 2
(Leucine + Arginine) bias of “dual topology” proteins
CrcB protein
family
YnfA protein
family
YdgC protein
family
(Leusine + Arginine) bias of “dual topology” proteins
YdgQ and YdgL
protein family
Not all proteins are dual topology proteins
One protein, two orientations in the membrane
DUF606
protein family
Most proteins 4 or 5 trans membrane helices.
Internally duplicated: 9 or 10 trans membrane helices
Internally duplicated protein DUF606
10 Trans Membrane
helices
N-terminus
36% sequence
identity
C terminus
Orientation of internally duplicated proteins
5 Trans membrane
helices protein
4 Trans membrane
helices protein
Internal duplication topology
Dual topology membrane proteins by different gene
Discussion

Dual topology proteins myth or reality ?
Discussion

Evolutionary path ?