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

Introduction Protein translocation into the mitochondria Introduction - Protein translocation into the mitochondria TOM TIM22 TIM23 Tom5 Tim12 Tim44 Tim14 Tim10 Tim mtHsp M. Sc. Mercedes RomeroAugust 07 SAM

Introduction Introduction - TOM complex Tom 22 Tom 40 Tom 6 Tom 5 Tom 7 Tom70 Tom22 Tom40 Tom20 Tom6 Tom5 Tom7 TOM holo complex M. Sc. Mercedes RomeroAugust 07 Tom70 Surface receptor Tom40 Translocation pore Tom22 Surface receptor Tom20 Surface receptor Tom5, 6 and 7 Stabilize the complex

Introduction Introduction - TOM complex Tom22 Tom40 Tom6 Tom5 Tom7 TOM core complex M. Sc. Mercedes RomeroAugust 07 Tom70 Surface receptor Tom40 Translocation pore Tom22 Surface receptor Tom20 Surface receptor Tom5, 6 and 7 Stablize the complex

I t 125mM KCl 10mM HEPES/KOH pH pA P = Ac-(AAKAA) 5 Y-NH 2 F M. Sc. Mercedes RomeroAugust 07 P Materials and methods - Electrophysiological experiments cistrans +

Materials and methods - Electrophysiological experiments I t 125mM KCl 10mM HEPES/KOH pH pA P F M. Sc. Mercedes RomeroAugust 07 P = Ac-(AAKAA) 5 Y-NH 2 cistrans +

Results – Single channel conductance of TOM core complex in the absence and presence of Results – Single channel conductance of TOM core complex in the absence and presence of Ac-(AAKAA) 5 Y-NH 2 M. Sc. Mercedes RomeroAugust 07 TOM core complex TOM core complex µM Ac-(AAKAA) 5 Y-NH 2 TOM core complex TOM core complex µM Ac-(AAKAA) 5 Y-NH 2 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

Results – TOM core complex open probabilities in the presence of diverse Ac-(AAKAA) 5 Y-NH 2 concentrations Ac-(AAKAA) 5 Y-NH 2 peptides reduce the channel open state probability not only in a voltage- also in a concentration-dependent manner. TOM core complex µM Ac-(AAKAA) 5 Y-NH 2 TOM core complex TOM core complex µM Ac-(AAKAA) 5 Y-NH 2

Results – Dose response curve M. Sc. Mercedes RomeroAugust 07 Estimation of a residency time of ca. 2 ms (*) (*) Hilles residency time formula [15-1], 1991

Looking into the future Fitting of the voltage dependence with a Woodhull model verification that the Ac-(AAKAA) 5 Y –NH 2 as an open channel blocker in the case of TOM complex position of the binding site Analysis of the kinetic parameters k on, k off 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 barrier M. Sc. Mercedes RomeroAugust 07

THANK YOU!!! THANK YOU!!! M. Sc. Mercedes RomeroAugust 07