Single protein molecules threading through the protein translocase in the mitochondrial outer membrane (TOM complex) M. Sc. Mercedes RomeroAugust 07.

Single protein molecules threading through the protein translocase in the

mitochondrial outer membrane

(TOM complex)

M. Sc. Mercedes Romero

August ‘07

IntroductionIntroduction - Protein translocation Protein translocation into the into the mitochondria mitochondria

TOM

TIM22

TIM23

Tom5

Tim12

Tim44

Tim14

Tim10Tim9

20 22

6 740

40

70

50

50

17

23-

mtHsp70

54

18

22

M. Sc. Mercedes Romero

August ‘07

SAM

IntroductionIntroduction - TOM complex

Tom 22

Tom 40

To

m 6

To

m 5

To

m 7

Tom70Tom22

Tom40

Tom20

To

m6

To

m5

To

m7

TOM holo complex

M. Sc. Mercedes Romero

August ‘07

Tom70Surface receptor Tom40Translocation pore Tom22Surface receptor Tom20Surface receptor Tom5, 6 and 7Stabilize the

complex

IntroductionIntroduction - TOM complex

Tom22

Tom40

To

m6

To

m5

To

m7

TOM core complex

M. Sc. Mercedes Romero

August ‘07

Tom70Surface receptor Tom40Translocation pore Tom22Surface receptor Tom20Surface receptor Tom5, 6 and 7Stablize the

complex

I

t125mM KCl 10mM HEPES/KOH pH 7.2

0 pA

P

= Ac-(AAKAA)5Y-NH2

F

M. Sc. Mercedes Romero

August ‘07

P

Materials and methodsMaterials and methods - - Electrophysiological Electrophysiological experimentsexperiments

cis trans+

Materials and methodsMaterials and methods - - Electrophysiological Electrophysiological experimentsexperiments

I

t125mM KCl 10mM HEPES/KOH pH 7.2

0 pA

P

F

M. Sc. Mercedes Romero

August ‘07

P= Ac-(AAKAA)5Y-NH2

cis trans+

ResultsResults – Single channel conductance of – Single channel conductance of TOM core complex in the absence and TOM core complex in the absence and presence of presence of Ac-(AAKAA)5Y-NH2

M. Sc. Mercedes Romero

August ‘07

-150 -100 -50 0 50 100 1500,0

0,2

0,4

0,6

0,8

1,0

Voltage/mV

Gno

rm

-150 -100 -50 0 50 100 1500,0

0,2

0,4

0,6

0,8

1,0

Voltage/mV

Gno

rm

TOM core complex

TOM core complex +0.67 µM Ac-(AAKAA)5Y-NH2

TOM core complex

TOM core complex +0.67 µM Ac-(AAKAA)5Y-

NH2

Normalized maximum conductance

• Main conductance state of 0.3 nS (at 125 mM KCl 10mM HEPES pH7.2)

• Rapid transitions to lower conductance levels at V>|40| mV. TOM core complex kinetic model multiple subconductance states.

Ac-(AAKAA)5Y-NH2

• Cationic alanine-based peptide

• Added into the trans chamber, where the positive electrode is placed

ResultsResults –– TOM core complex open TOM core complex open probabilities in the presence of diverse Ac-probabilities in the presence of diverse Ac-

(AAKAA)(AAKAA)55Y-NHY-NH22 concentrations concentrations

Voltage/mV

P0

-150 -100 -50 0 50 100 1500,0

0,2

0,4

0,6

0,8

1,0

VV

right

left

AA

rightleft

Voltage/mV

-150 -100 -50 0 50 100 1500,0

0,2

0,4

0,6

0,8

1,0

P0V

right

Voltage/mV

-150 -100 -50 0 50 100 1500,0

0,2

0,4

0,6

0,8

1,0

P0V

right

)))(*exp(1(

1(*)))(*exp(1

1(0rightVV

rightA

leftVV

leftA

P

Ac-(AAKAA)5Y-NH2 peptides reduce the channel open state probability not only in a voltage- also in a concentration-

dependent manner.

TOM core complex +0.67 µM Ac-(AAKAA)5Y-NH2

TOM core complex

TOM core complex +1.43 µM Ac-(AAKAA)5Y-NH2

ResultsResults – Dose response curve – Dose response curve

M. Sc. Mercedes Romero

August ‘07

Kd = 0.5 µM

1E-8 1E-7 1E-6 1E-5 1E-4 1E-3 0,01 0,1 1 100

20

40

60

80

100

V/m

Vri

gh

t

Ac-(AAKAA) Y-NH concentration / M5 2

[Ac-(AAKAA) Y-NH ] (µM)5 2

0.000.010.120.230.300.671.432.75

V (mV)right

59.870.070.062.565.030.025.027.6

±SD (mV)

± 5.9± 5.5± 9.4± 3.2± 9.1± 3.4± 6.0± 2.4

Estimation of a residency time of ca. 2

ms (*)

(*) Hille‘s residency time formula [15-1], 1991

Looking into the futureLooking into the future

Fitting of the voltage dependence with a Woodhull model verification that the Ac-(AAKAA)5Y –NH2 as

an open channel blocker in the case of TOM complex position of the binding site

Analysis of the kinetic parameters kon, koff partitioning coefficient

Peptide translocation experiments Tom 40 (the core unit)

to avoid the TOM complex voltage dependence

gating

Study of the contribution of the peptide charge peptide length to the

free energy barrierM. Sc. Mercedes Romero

August ‘07

THANK YOU!!!THANK YOU!!!

M. Sc. Mercedes Romero

August ‘07