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