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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