Melissa DavidAdam OssinRutger Mantingh
Supervisor:Antoinette Killian
Periplasm
Cytoplasm
Inner membrane
IntroductionIntegral 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
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
Protein 1 Protein 2 Protein 1 Protein 2
Singleton Gene pair
Protein 1 Protein 2 Protein 1 Protein 2
Singleton Gene pair
Protein 1 Protein 2 Protein 1 Protein 2
Singleton Gene pair
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 C-terminus: PhoA: enzymatically active
only in the preiplasm
GFP: florescent only in cytoplasm
PhoA
GFP PhoA
GFP
Methods (2) How to determine
biases? Unbiased proteins
are incorporated either way (dual-topology)
Biased proteins are incorporated in one way
Methods (3) MutationsAddition or
substitution of/with positive amino acids (K + R)
SugE and EmrE
EmrE & SugE
SugE and EmrE
EmrE & SugE
PhoA
GFP PhoA
GFP
SugE and EmrE
EmrE & SugE
PhoA
GFP PhoA
GFP
ControlYdgE YdgF
CrcB
CrcB
YnfA and YdgCYnfA YdgC
Dual topology proteins: A single gene or a gene pair
Protein 1 Protein 2 Protein 1 Protein 2
Singleton Gene pair
Protein 1 Protein 2 Protein 1 Protein 2
Singleton Gene pair
225 fully sequenced genomes scanned for pairs and singletons
SMR protein family -Both singletons and gene pairs-Singletons around (K+R) bias = 0-Gene pairs bigger (K+R) bias
Determination by :
(Leucine + Arginine) bias of “dual topology” proteins
Protein 1 Protein 2 Protein 1 Protein 2
Singleton Gene pair
SMR proteinfamily
CrcB proteinfamily
YnfA proteinfamily
YdgC proteinfamily
(Leucine + Arginine) bias of “dual topology” proteins
YdgQ and YdgLprotein family
Not all proteins are dual topology proteins
(Leusine + Arginine) bias of “dual topology” proteins
One protein, two orientations in the membrane
DUF606protein family
Most proteins 4 or 5 trans membrane helices.
Internally duplicated: 9 or 10 trans membrane helices
Internally duplicated protein DUF606
36% sequence identity
10 Trans Membranehelices
N-terminus 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
Dual topology proteins myth or reality ?
Discussion
Evolutionary path ?
Discussion